Optimising T cell (re)boosting strategies for adenoviral and modified vaccinia Ankara vaccine regimens in humans.

Simian adenoviral and modified vaccinia Ankara (MVA) viral vectors used in heterologous prime-boost strategies are potent inducers of T cells against encoded antigens and are in advanced testing as vaccine carriers for a wide range of infectious agents and cancers. It is unclear if these responses can be further enhanced or sustained with reboosting strategies. Furthermore, despite the challenges involved in MVA manufacture dose de-escalation has not been performed in humans. In this study, healthy volunteers received chimpanzee-derived adenovirus-3 and MVA vaccines encoding the non-structural region of hepatitis C virus (ChAd3-NSmut/MVA-NSmut) 8 weeks apart. Volunteers were then reboosted with a second round of ChAd3-NSmut/MVA-NSmut or MVA-NSmut vaccines 8 weeks or 1-year later. We also determined the capacity of reduced doses of MVA-NSmut to boost ChAd3-NSmut primed T cells. Reboosting was safe, with no enhanced reactogenicity. Reboosting after an 8-week interval led to minimal re-expansion of transgene-specific T cells. However, after a longer interval, T cell responses expanded efficiently and memory responses were enhanced. The 8-week interval regimen induced a higher percentage of terminally differentiated and effector memory T cells. Reboosting with MVA-NSmut alone was as effective as with ChAd3-NSmut/MVA-NSmut. A ten-fold lower dose of MVA (2 × 107pfu) induced high-magnitude, sustained, broad, and functional Hepatitis C virus (HCV)-specific T cell responses, equivalent to standard doses (2 × 108 pfu). Overall, we show that following Ad/MVA prime-boost vaccination reboosting is most effective after a prolonged interval and is productive with MVA alone. Importantly, we also show that a ten-fold lower dose of MVA is as potent in humans as the standard dose.

Rapid Affinity Maturation of Novel Anti-PD-L1 Antibodies by a Fast Drop of the Antigen Concentration and FACS Selection of Yeast Libraries.

The affinity engineering is a key step to increase the efficacy of therapeutic monoclonal antibodies and yeast surface display is the most widely used and powerful affinity maturation approach, achieving picomolar binding affinities. In this study, we provide an optimization of the yeast surface display methodology, applied to the generation of potentially therapeutic high affinity antibodies targeting the immune checkpoint PD-L1. In this approach, we coupled a 10-cycle error-prone mutagenesis of heavy chain complementarity determining region 3 of an anti-PD-L1 scFv, previously identified by phage display, with high-throughput sequencing, to generate scFv-yeast libraries with high mutant frequency and diversity. In addition, we set up a novel, faster and effective selection scheme by fluorescence-activated cell sorting, based on a fast drop of the antigen concentration between the first and the last selection cycles, unlike the gradual decrease typical of current selection protocols. In this way we isolated 6 enriched mutated scFv-yeast clones overall, showing an affinity improvement for soluble PD-L1 protein compared to the parental scFv. As a proof of the potency of the novel approach, we confirmed that the antibodies converted from all the mutated scFvs retained the affinity improvement. Remarkably, the best PD-L1 binder among them also bound with a higher affinity to PD-L1 expressed in its native conformation on human-activated lymphocytes, and it was able to stimulate lymphocyte proliferation in vitro more efficiently than its parental antibody. This optimized technology, besides the identification of a new potential checkpoint inhibitor, provides a tool for the quick isolation of high affinity binders.

Massive parallel screening of phage libraries for the generation of repertoires of human immunomodulatory monoclonal antibodies.

Immune checkpoints are emerging as novel targets for cancer therapy, and antibodies against them have shown remarkable clinical efficacy with potential for combination treatments to achieve high therapeutic index. This work aims at providing a novel approach for the generation of several novel human immunomodulatory antibodies capable of binding their targets in their native conformation and useful for therapeutic applications. We performed a massive parallel screening of phage libraries by using for the first time activated human lymphocytes to generate large collections of single-chain variable fragments (scFvs) against 10 different immune checkpoints: LAG-3, PD-L1, PD-1, TIM3, BTLA, TIGIT, OX40, 4-1BB, CD27 and ICOS. By next-generation sequencing and bioinformatics analysis we ranked individual scFvs in each collection and identified those with the highest level of enrichment. As a proof of concept of the quality/potency of the binders identified by this approach, human IgGs from three of these collections (i.e., PD-1, PD-L1 and LAG-3) were generated and shown to have comparable or better binding affinity and biological activity than the clinically validated anti-PD-1 mAb nivolumab. The repertoires generated in this work represent a convenient source of agonistic or antagonistic antibodies against the 'Checkpoint Immunome' for preclinical screening and clinical implementation of optimized treatments.

Incidence and Risk Factors for Hepatitis C Virus Infection among Illicit Drug Users in Italy.

So far, only three small outdated studies have investigated hepatitis C virus (HCV) incidence and risk factors among illicit drug users (DUs) in Italy. Thus, during 2007-2010, we conducted a prospective cohort study among DUs attending 17 Italian rehabilitation centers serving urban areas. Two hundred eighty-four HCV-uninfected DUs were prospectively followed by interview and anti-HCV antibody and RNA testing every 6 months. Incidence was calculated using the person-years method. Infection predictors were assessed by time-dependent Cox analysis. Participants were mostly male (83.4%), under opioid substitution therapy (OST) (78.9%), non-injecting DUs (67.9%), and with a mean age of 30.8. Ninety-one of 224 DUs initially under OST interrupted treatment during the follow-up. Overall HCV incidence was 5.83/100 person-years at risk (PYAR) [95% confidence intervals (CI), 3.63-9.38]. The incidence did not significantly differ according the participants' sociodemographic characteristics or the degree of urbanization of the towns involved in the study. The incidence was higher for DUs under than for those not under OST (6.23 vs 4.50/100 PYAR; p = 0.681). Incidence was also higher for those with than for those without OST interruption (7.17 vs 5.04/100 PYAR; p = 0.55). However, all these differences were non-significant. At last follow-up visit, a significant decrease in frequency of sharing equipment for preparation/using drugs (by injection or not) was observed by analyzing either the whole cohort or DUs under OST only. Anti-HCV seroconversion resulted independently associated with sharing drug preparation/use equipment, backloading, having a HCV-positive sexual partner, or household and (marginally) intravenous injection. In this study, HCV incidence was non-negligible and OST seemed to lack effectiveness in reducing it. In Italy, implementation of combined harm reduction interventions and antiviral treatment of chronically infected DUs would be needed.

Safety and Immunogenicity of Malaria Vectored Vaccines Given with Routine Expanded Program on Immunization Vaccines in Gambian Infants and Neonates: A Randomized Controlled Trial.

BACKGROUND: Heterologous prime-boost vaccination with chimpanzee adenovirus 63 (ChAd63) and modified vaccinia virus Ankara (MVA) encoding multiple epitope string thrombospondin-related adhesion protein (ME-TRAP) has shown acceptable safety and promising immunogenicity in African adult and pediatric populations. If licensed, this vaccine could be given to infants receiving routine childhood immunizations. We therefore evaluated responses to ChAd63 MVA ME-TRAP when co-administered with routine Expanded Program on Immunization (EPI) vaccines. METHODS: We enrolled 65 Gambian infants and neonates, aged 16, 8, or 1 week at first vaccination and randomized them to receive either ME-TRAP and EPI vaccines or EPI vaccines only. Safety was assessed by the description of vaccine-related adverse events (AEs). Immunogenicity was evaluated using IFNγ enzyme-linked immunospot, whole-blood flow cytometry, and anti-TRAP IgG ELISA. Serology was performed to confirm all infants achieved protective titers to EPI vaccines. RESULTS: The vaccines were well tolerated in all age groups with no vaccine-related serious AEs. High-level TRAP-specific IgG and T cell responses were generated after boosting with MVA. CD8+ T cell responses, previously found to correlate with protection, were induced in all groups. Antibody responses to EPI vaccines were not altered significantly. CONCLUSION: Malaria vectored prime-boost vaccines co-administered with routine childhood immunizations were well tolerated. Potent humoral and cellular immunity induced by ChAd63 MVA ME-TRAP did not reduce the immunogenicity of co-administered EPI vaccines, supporting further evaluation of this regimen in infant populations. CLINICAL TRIAL REGISTRATION: The clinical trial was registered on http://Clinicaltrials.gov (NCT02083887) and the Pan-African Clinical Trials Registry (PACTR201402000749217).

Functional mapping of brain synapses by the enriching activity-marker SynaptoZip.

Ideally, elucidating the role of specific brain circuits in animal behavior would require the ability to measure activity at all involved synapses, possibly with unrestricted field of view, thus even at those boutons deeply located into the brain. Here, we introduce and validate an efficient scheme reporting synaptic vesicle cycling in vivo. This is based on SynaptoZip, a genetically encoded molecule deploying in the vesicular lumen a bait moiety designed to capture upon exocytosis a labeled alien peptide, Synbond. The resulting signal is cumulative and stores the number of cycling events occurring at individual synapses. Since this functional signal is enduring and measurable both online and ex post, SynaptoZip provides a unique method for the analysis of the history of synaptic activity in regions several millimeters below the brain surface. We show its broad applicability by reporting stimulus-evoked and spontaneous circuit activity in wide cortical fields, in anesthetized and freely moving animals.

A first-in-human study of the novel HIV-fusion inhibitor C34-PEG4-Chol.

Long-acting injectable antiretroviral (LA-ARV) drugs with low toxicity profiles and propensity for drug-drug interactions are a goal for future ARV regimens. C34-PEG4-Chol is a novel cholesterol tagged LA HIV-fusion-inhibitor (FI). We assessed pre-clinical toxicology and first-in-human administration of C34-PEG4-Chol. Pre-clinical toxicology was conducted in 2 species. HIV-positive men were randomised to a single subcutaneous dose of C34-PEG4-Chol at incrementing doses or placebo. Detailed clinical (including injection site reaction (ISR) grading), plasma pharmacokinetic (time-to-minimum-effective-concentration (MEC, 25 ng/mL) and pharmacodynamic (plasma HIV RNA) parameters were assessed. In both mice and dogs, no-observed-adverse effect level (NOAEL) was observed at a 12 mg/kg/dose after two weeks. Of 5 men enrolled, 3 received active drug (10 mg, 10 mg and 20 mg). In 2 individuals grade 3 ISR occurred and the study was halted. Both ISR emerged within 12 hours of active drug dosing. No systemic toxicities were observed. The time-to-MEC was >72 and >96 hours after 10 and 20 mg dose, respectively, and mean change in HIV RNA was -0.9 log10 copies/mL. These human pharmacodynamic and pharmacokinetic data, although limited to 3 subjects, of C34-PEG-4-Chol suggest continuing evaluation of this agent as a LA-ARV. However, alternative administration routes must be explored.

A third generation vaccine for human visceral leishmaniasis and post kala azar dermal leishmaniasis: First-in-human trial of ChAd63-KH.

BACKGROUND: Visceral leishmaniasis (VL or kala azar) is the most serious form of human leishmaniasis, responsible for over 20,000 deaths annually, and post kala azar dermal leishmaniasis (PKDL) is a stigmatizing skin condition that often occurs in patients after successful treatment for VL. Lack of effective or appropriately targeted cell mediated immunity, including CD8+ T cell responses, underlies the progression of VL and progression to PKDL, and can limit the therapeutic efficacy of anti-leishmanial drugs. Hence, in addition to the need for prophylactic vaccines against leishmaniasis, the development of therapeutic vaccines for use alone or in combined immuno-chemotherapy has been identified as an unmet clinical need. Here, we report the first clinical trial of a third-generation leishmaniasis vaccine, developed intentionally to induce Leishmania-specific CD8+ T cells. METHODS: We conducted a first-in-human dose escalation Phase I trial in 20 healthy volunteers to assess the safety, tolerability and immunogenicity of a prime-only adenoviral vaccine for human VL and PKDL. ChAd63-KH is a replication defective simian adenovirus expressing a novel synthetic gene (KH) encoding two Leishmania proteins KMP-11 and HASPB. Uniquely, the latter was engineered to reflect repeat domain polymorphisms and arrangements identified from clinical isolates. We monitored innate immune responses by whole blood RNA-Seq and antigen specific CD8+ T cell responses by IFNγ ELISPOT and intracellular flow cytometry. FINDINGS: ChAd63-KH was safe at intramuscular doses of 1x1010 and 7.5x1010 vp. Whole blood transcriptomic profiling indicated that ChAd63-KH induced innate immune responses characterized by an interferon signature and the presence of activated dendritic cells. Broad and quantitatively robust CD8+ T cell responses were induced by vaccination in 100% (20/20) of vaccinated subjects. CONCLUSION: The results of this study support the further development of ChAd63-KH as a novel third generation vaccine for VL and PKDL. TRIAL REGISTRATION: This clinical trial (LEISH1) was registered at EudraCT (2012-005596-14) and ISRCTN (07766359).

Mucosal Vaccination with Heterologous Viral Vectored Vaccine Targeting Subdominant SIV Accessory Antigens Strongly Inhibits Early Viral Replication.

Conventional HIV T cell vaccine strategies have not been successful in containing acute peak viremia, nor in providing long-term control. We immunized rhesus macaques intramuscularly and rectally using a heterologous adenovirus vectored SIV vaccine regimen encoding normally weakly immunogenic tat, vif, rev and vpr antigens fused to the MHC class II associated invariant chain. Immunizations induced broad T cell responses in all vaccinees. Following up to 10 repeated low-dose intrarectal challenges, vaccinees suppressed early viral replication (P=0.01) and prevented the peak viremia in 5/6 animals. Despite consistently undetectable viremia in 2 out of 6 vaccinees, all animals showed evidence of infection induced immune responses indicating that infection had taken place. Vaccinees, with and without detectable viremia better preserved their rectal CD4+ T cell population and had reduced immune hyperactivation as measured by naïve T cell depletion, Ki-67 and PD-1 expression on T cells. These results indicate that vaccination towards SIV accessory antigens vaccine can provide a level of acute control of SIV replication with a suggestion of beneficial immunological consequences in infected animals of unknown long-term significance. In conclusion, our studies demonstrate that a vaccine encoding subdominant antigens not normally associated with virus control can exert a significant impact on acute peak viremia.

Therapeutic Vaccine Against Primate Papillomavirus Infections of the Cervix.

Currently available prophylactic vaccines have no therapeutic efficacy for preexisting human papillomavirus (HPVs) infections, do not target all oncogenic HPVs and are insufficient to eliminate the burden of HPV induced cancer. We aim to develop an alternative HPV vaccine which is broadly effective and capable of clearing preexisting infection. In an initial attempt to develop a broadly reactive therapeutic vaccine, we designed a putative papillomavirus (PV) ancestor antigen (circulating sequence derived antigenic sequences E1E2-CDSE1E2) based on the conserved E1 and E2 protein sequences from existing oncogenic HPV strains. This antigen was found to be as related to circulating oncogenic Macaca fascicularis papillomaviruses (MfPVs) as to oncogenic HPVs. The CDSE1E2 antigen was fused to a T-cell adjuvant and encoded in chimpanzee 3 and 63 adenoviral vectors. We first showed that the combination of these 2 vaccines induced long-lasting potent CDSE1E2 specific T cell responses in outbred mice. This prime-boost regimen was then tested in female macaques naturally infected with MfPVs. All immunized animals (16/16) responded to the vaccine antigen but with reduced cross-reactivity against existing PVs. Preexisting MfPV infections did not prime vaccine inducible immune responses. Importantly, immunized oncogenic MfPV type 3 (MfPV3) infected animals that responded toward MfPV3 were able to diminish cervical MfPV3 DNA content. Although insufficient breadth was achieved, our results suggest that a relevant level of E1E2 specific T cell immunity is achievable and might be sufficient for the elimination of PV infection. Importantly, naturally infected macaques, offer a relevant model for testing vaccines aimed at eliminating mucosal PV infections.

Viral Vector Malaria Vaccines Induce High-Level T Cell and Antibody Responses in West African Children and Infants.

Heterologous prime-boosting with viral vectors encoding the pre-erythrocytic antigen thrombospondin-related adhesion protein fused to a multiple epitope string (ME-TRAP) induces CD8+ T cell-mediated immunity to malaria sporozoite challenge in European malaria-naive and Kenyan semi-immune adults. This approach has yet to be evaluated in children and infants. We assessed this vaccine strategy among 138 Gambian and Burkinabe children in four cohorts: 2- to 6-year olds in The Gambia, 5- to 17-month-olds in Burkina Faso, and 5- to 12-month-olds and 10-week-olds in The Gambia. We assessed induction of cellular immunity, taking into account the distinctive hematological status of young infants, and characterized the antibody response to vaccination. T cell responses peaked 7 days after boosting with modified vaccinia virus Ankara (MVA), with highest responses in infants aged 10 weeks at priming. Incorporating lymphocyte count into the calculation of T cell responses facilitated a more physiologically relevant comparison of cellular immunity across different age groups. Both CD8+ and CD4+ T cells secreted cytokines. Induced antibodies were up to 20-fold higher in all groups compared with Gambian and United Kingdom (UK) adults, with comparable or higher avidity. This immunization regimen elicited strong immune responses, particularly in young infants, supporting future evaluation of efficacy in this key target age group for a malaria vaccine.

Chimpanzee Adenovirus Vector Ebola Vaccine.

BACKGROUND: The unprecedented 2014 epidemic of Ebola virus disease (EVD) prompted an international response to accelerate the availability of a preventive vaccine. A replication-defective recombinant chimpanzee adenovirus type 3-vectored ebolavirus vaccine (cAd3-EBO), encoding the glycoprotein from Zaire and Sudan species, that offers protection in the nonhuman primate model, was rapidly advanced into phase 1 clinical evaluation. METHODS: We conducted a phase 1, dose-escalation, open-label trial of cAd3-EBO. Twenty healthy adults, in sequentially enrolled groups of 10 each, received vaccination intramuscularly in doses of 2×1010 particle units or 2×1011 particle units. Primary and secondary end points related to safety and immunogenicity were assessed throughout the first 8 weeks after vaccination; in addition, longer-term vaccine durability was assessed at 48 weeks after vaccination. RESULTS: In this small study, no safety concerns were identified; however, transient fever developed within 1 day after vaccination in two participants who had received the 2×1011 particle-unit dose. Glycoprotein-specific antibodies were induced in all 20 participants; the titers were of greater magnitude in the group that received the 2×1011 particle-unit dose than in the group that received the 2×1010 particle-unit dose (geometric mean titer against the Zaire antigen at week 4, 2037 vs. 331; P=0.001). Glycoprotein-specific T-cell responses were more frequent among those who received the 2×1011 particle-unit dose than among those who received the 2×1010 particle-unit dose, with a CD4 response in 10 of 10 participants versus 3 of 10 participants (P=0.004) and a CD8 response in 7 of 10 participants versus 2 of 10 participants (P=0.07) at week 4. Assessment of the durability of the antibody response showed that titers remained high at week 48, with the highest titers in those who received the 2×1011 particle-unit dose. CONCLUSIONS: Reactogenicity and immune responses to cAd3-EBO vaccine were dose-dependent. At the 2×1011 particle-unit dose, glycoprotein Zaire-specific antibody responses were in the range reported to be associated with vaccine-induced protective immunity in challenge studies involving nonhuman primates, and responses were sustained to week 48. Phase 2 studies and efficacy trials assessing cAd3-EBO are in progress. (Funded by the Intramural Research Program of the National Institutes of Health; VRC 207 ClinicalTrials.gov number, NCT02231866 .).

Highly-Immunogenic Virally-Vectored T-cell Vaccines Cannot Overcome Subversion of the T-cell Response by HCV during Chronic Infection.

An effective therapeutic vaccine for the treatment of chronic hepatitis C virus (HCV) infection, as an adjunct to newly developed directly-acting antivirals (DAA), or for the prevention of reinfection, would significantly reduce the global burden of disease associated with chronic HCV infection. A recombinant chimpanzee adenoviral (ChAd3) vector and a modified vaccinia Ankara (MVA), encoding the non-structural proteins of HCV (NSmut), used in a heterologous prime/boost regimen induced multi-specific, high-magnitude, durable HCV-specific CD4+ and CD8+ T-cell responses in healthy volunteers, and was more immunogenic than a heterologous Ad regimen. We now assess the immunogenicity of this vaccine regimen in HCV infected patients (including patients with a low viral load suppressed with interferon/ribavirin therapy), determine T-cell cross-reactivity to endogenous virus, and compare immunogenicity with that observed previously in both healthy volunteers and in HCV infected patients vaccinated with the heterologous Ad regimen. Vaccination of HCV infected patients with ChAd3-NSmut/MVA-NSmut was well tolerated. Vaccine-induced HCV-specific T-cell responses were detected in 8/12 patients; however, CD4+ T-cell responses were rarely detected, and the overall magnitude of HCV-specific T-cell responses was markedly reduced when compared to vaccinated healthy volunteers. Furthermore, HCV-specific cells had a distinct partially-functional phenotype (lower expression of activation markers, granzyme B, and TNFα production, weaker in vitro proliferation, and higher Tim3 expression, with comparable Tbet and Eomes expression) compared to healthy volunteers. Robust anti-vector T-cells and antibodies were induced, showing that there is no global defect in immunity. The level of viremia at the time of vaccination did not correlate with the magnitude of the vaccine-induced T-cell response. Full-length, next-generation sequencing of the circulating virus demonstrated that T-cells were only induced by vaccination when there was a sequence mismatch between the autologous virus and the vaccine immunogen. However, these T-cells were not cross-reactive with the endogenous viral variant epitopes. Conversely, when there was complete homology between the immunogen and circulating virus at a given epitope T-cells were not induced. T-cell induction following vaccination had no significant impact on HCV viral load. In vitro T-cell culture experiments identified the presence of T-cells at baseline that could be expanded by vaccination; thus, HCV-specific T-cells may have been expanded from pre-existing low-level memory T-cell populations that had been exposed to HCV antigens during natural infection, explaining the partial T-cell dysfunction. In conclusion, vaccination with ChAd3-NSmut and MVA-NSmut prime/boost, a potent vaccine regimen previously optimized in healthy volunteers was unable to reconstitute HCV-specific T-cell immunity in HCV infected patients. This highlights the major challenge of overcoming T-cell exhaustion in the context of persistent antigen exposure.

Safety and Immunogenicity of ChAd63 and MVA ME-TRAP in West African Children and Infants.

Malaria remains a significant global health burden and a vaccine would make a substantial contribution to malaria control. Chimpanzee Adenovirus 63 Modified Vaccinia Ankara Multiple epitope thrombospondin adhesion protein (ME-TRAP) and vaccination has shown significant efficacy against malaria sporozoite challenge in malaria-naive European volunteers and against malaria infection in Kenyan adults. Infants are the target age group for malaria vaccination; however, no studies have yet assessed T-cell responses in children and infants. We enrolled 138 Gambian and Burkinabe children in four different age-groups: 2-6 years old in The Gambia; 5-17 months old in Burkina Faso; 5-12 months old, and also 10 weeks old, in The Gambia; and evaluated the safety and immunogenicity of Chimpanzee Adenovirus 63 Modified Vaccinia Ankara ME-TRAP heterologous prime-boost immunization. The vaccines were well tolerated in all age groups with no vaccine-related serious adverse events. T-cell responses to vaccination peaked 7 days after boosting with Modified Vaccinia Ankara, with T-cell responses highest in 10 week-old infants. Heterologous prime-boost immunization with Chimpanzee Adenovirus 63 and Modified Vaccinia Ankara ME-TRAP was well tolerated in infants and children, inducing strong T-cell responses. We identify an approach that induces potent T-cell responses in infants, which may be useful for preventing other infectious diseases requiring cellular immunity.

Antibody response to respiratory syncytial virus infection in children <18 months old.

The development of a safe and effective respiratory syncytial virus (RSV) vaccine might be facilitated by knowledge of the natural immune response to this virus. The aims of this study were to evaluate the neutralizing antibody response of a cohort of healthy children <18 months old to RSV infection. During the RSV season, 89 healthy children <18 months old were enrolled and followed up weekly for 12 weeks. At each visit, a nasopharyngeal swab was obtained for RSV detection by real-time polymerase chain reaction (PCR). During the study period, 2 blood samples were drawn and they were used to determine RSV geometric mean neutralizing antibody titres (GMT) against RSV. A total of 35 (39.3%) children had RSV detected during the study period. Among RSV-positive patients, children ≥7 months showed a significantly higher increase in antibody response (p<0.001). A significantly higher number of patients with a ≥4 -fold increase in GMT were ≥7 months old (p = 0.02) and presented lower respiratory tract infections (LRTIs) during the study period (p = 0.01). Viral shedding was longer among children aged ≥7 months (p = 0.06), those with viral load ≥10(6) copies/mL (p = 0.03), and those with LRTIs during the study period (p = 0.03), but it was not associated with the immune response (p = 0.41). In conclusion, natural RSV infection seems to evoke a low immune response in younger children. To be effective in this infant population, which is at highest risk of developing severe LRTIs, vaccines must be able to induce in the first months of life a stronger immune response than that produced by the natural infection.

A Monovalent Chimpanzee Adenovirus Ebola Vaccine Boosted with MVA.

BACKGROUND: The West African outbreak of Ebola virus disease that peaked in 2014 has caused more than 11,000 deaths. The development of an effective Ebola vaccine is a priority for control of a future outbreak. METHODS: In this phase 1 study, we administered a single dose of the chimpanzee adenovirus 3 (ChAd3) vaccine encoding the surface glycoprotein of Zaire ebolavirus (ZEBOV) to 60 healthy adult volunteers in Oxford, United Kingdom. The vaccine was administered in three dose levels--1×10(10) viral particles, 2.5×10(10) viral particles, and 5×10(10) viral particles--with 20 participants in each group. We then assessed the effect of adding a booster dose of a modified vaccinia Ankara (MVA) strain, encoding the same Ebola virus glycoprotein, in 30 of the 60 participants and evaluated a reduced prime-boost interval in another 16 participants. We also compared antibody responses to inactivated whole Ebola virus virions and neutralizing antibody activity with those observed in phase 1 studies of a recombinant vesicular stomatitis virus-based vaccine expressing a ZEBOV glycoprotein (rVSV-ZEBOV) to determine relative potency and assess durability. RESULTS: No safety concerns were identified at any of the dose levels studied. Four weeks after immunization with the ChAd3 vaccine, ZEBOV-specific antibody responses were similar to those induced by rVSV-ZEBOV vaccination, with a geometric mean titer of 752 and 921, respectively. ZEBOV neutralization activity was also similar with the two vaccines (geometric mean titer, 14.9 and 22.2, respectively). Boosting with the MVA vector increased virus-specific antibodies by a factor of 12 (geometric mean titer, 9007) and increased glycoprotein-specific CD8+ T cells by a factor of 5. Significant increases in neutralizing antibodies were seen after boosting in all 30 participants (geometric mean titer, 139; P<0.001). Virus-specific antibody responses in participants primed with ChAd3 remained positive 6 months after vaccination (geometric mean titer, 758) but were significantly higher in those who had received the MVA booster (geometric mean titer, 1750; P<0.001). CONCLUSIONS: The ChAd3 vaccine boosted with MVA elicited B-cell and T-cell immune responses to ZEBOV that were superior to those induced by the ChAd3 vaccine alone. (Funded by the Wellcome Trust and others; ClinicalTrials.gov number, NCT02240875.).

Targeting a host-cell entry factor barricades antiviral-resistant HCV variants from on-therapy breakthrough in human-liver mice.

OBJECTIVE: Direct-acting antivirals (DAAs) inhibit hepatitis C virus (HCV) infection by targeting viral proteins that play essential roles in the replication process. However, selection of resistance-associated variants (RAVs) during DAA therapy has been a cause of therapeutic failure. In this study, we wished to address whether such RAVs could be controlled by the co-administration of host-targeting entry inhibitors that prevent intrahepatic viral spread. DESIGN: We investigated the effect of adding an entry inhibitor (the anti-scavenger receptor class B type I mAb1671) to a DAA monotherapy (the protease inhibitor ciluprevir) in human-liver mice chronically infected with HCV of genotype 1b. Clinically relevant non-laboratory strains were used to achieve viraemia consisting of a cloud of related viral variants (quasispecies) and the emergence of RAVs was monitored at high resolution using next-generation sequencing. RESULTS: HCV-infected human-liver mice receiving DAA monotherapy rapidly experienced on-therapy viral breakthrough. Deep sequencing of the HCV protease domain confirmed the manifestation of drug-resistant mutants upon viral rebound. In contrast, none of the mice treated with a combination of the DAA and the entry inhibitor experienced on-therapy viral breakthrough, despite detection of RAV emergence in some animals. CONCLUSIONS: This study provides preclinical in vivo evidence that addition of an entry inhibitor to an anti-HCV DAA regimen restricts the breakthrough of DAA-resistant viruses. Our approach is an excellent strategy to prevent therapeutic failure caused by on-therapy rebound of DAA-RAVs. Inclusion of an entry inhibitor to the newest DAA combination therapies may further increase response rates, especially in difficult-to-treat patient populations.

Novel human anti-claudin 1 mAbs inhibit hepatitis C virus infection and may synergize with anti-SRB1 mAb.

Hepatitis C virus (HCV) is a major cause of chronic hepatitis and liver carcinoma and new therapies based on novel targets are needed. The tight junction protein claudin 1 (CLDN-1) is essential for HCV cell entry and spread, and anti-CLDN-1 rat and mouse mAbs are safe and effective in preventing and treating HCV infection in a human liver chimeric mouse model. To accelerate translation of these observations into a novel approach to treat HCV infection and disease in humans, we screened a phage display library of human single-chain antibody fragments by using a panel of CLDN-1-positive and -negative cell lines and identified phage specifically binding to CLDN-1. The 12 clones showing the highest levels of binding were converted into human IgG4. Some of these mAbs displayed low-nanomolar affinity, and inhibited infection of human hepatoma Huh7.5 cells by different HCV isolates in a dose-dependent manner. Cross-competition experiments identified six inhibitory mAbs that recognized distinct epitopes. Combination of the human anti-SRB1 mAb C-1671 with these anti-CLDN-1 mAbs could either increase or reduce inhibition of cell culture-derived HCV infection in vitro. These novel human anti-CLDN-1 mAbs are potentially useful to develop a new strategy for anti-HCV therapy and lend support to the combined use of antibodies targeting the HCV receptors CLDN-1 and SRB1, but indicate that care must be taken in selecting the proper combination.

Chronic hepatitis C viral infection subverts vaccine-induced T-cell immunity in humans.

UNLABELLED: Adenoviral vectors encoding hepatitis C virus (HCV) nonstructural (NS) proteins induce multispecific, high-magnitude, durable CD4(+) and CD8(+) T-cell responses in healthy volunteers. We assessed the capacity of these vaccines to induce functional HCV-specific immune responses and determine T-cell cross-reactivity to endogenous virus in patients with chronic HCV infection. HCV genotype 1-infected patients were vaccinated using heterologous adenoviral vectors (ChAd3-NSmut and Ad6-NSmut) encoding HCV NS proteins in a dose escalation, prime-boost regimen, with and without concomitant pegylated interferon-α/ribavirin therapy. Analysis of immune responses ex vivo used human leukocyte antigen class I pentamers, intracellular cytokine staining, and fine mapping in interferon-γ enzyme-linked immunospot assays. Cross-reactivity of T cells with population and endogenous viral variants was determined following viral sequence analysis. Compared to healthy volunteers, the magnitude of HCV-specific T-cell responses following vaccination was markedly reduced. CD8(+) HCV-specific T-cell responses were detected in 15/24 patients at the highest dose, whereas CD4(+) T-cell responses were rarely detectable. Analysis of the host circulating viral sequence showed that T-cell responses were rarely elicited when there was sequence homology between vaccine immunogen and endogenous virus. In contrast, T cells were induced in the context of genetic mismatch between vaccine immunogen and endogenous virus; however, these commonly failed to recognize circulating epitope variants and had a distinct partially functional phenotype. Vaccination was well tolerated but had no significant effect on HCV viral load. CONCLUSION: Vaccination with potent HCV adenoviral vectored vaccines fails to restore T-cell immunity except where there is genetic mismatch between vaccine immunogen and endogenous virus; this highlights the major challenge of overcoming T-cell exhaustion in the context of persistent antigen exposure with implications for cancer and other persistent infections.

Persistent hepatitis C viral replication despite priming of functional CD8+ T cells by combined therapy with a vaccine and a direct-acting antiviral.

UNLABELLED: Exhaustion of antiviral CD8(+) T cells contributes to persistence of hepatitis C viral (HCV) infection. This immune response has proved difficult to restore by therapeutic vaccination, even when HCV replication is suppressed using antiviral regimens containing type I interferon. Because immunomodulatory effects of type I interferon may be a factor in poor T-cell priming, we undertook therapeutic vaccination in two chronically infected chimpanzees during treatment with a direct-acting antiviral (DAA) targeting the HCV NS5b polymerase protein. Immunization with genetic vaccines encoding the HCV NS3-NS5b nonstructural proteins during DAA treatment resulted in a multifunctional CD8(+) T-cell response. However, these antiviral CD8(+) T cells did not prevent persistent replication of DAA-resistant HCV variants that emerged during treatment. Most vaccine-induced CD8(+) T cells targeted class I epitopes that were not conserved in the circulating virus. Exhausted intrahepatic CD8(+) T-cell targeting-conserved epitopes did not expand after vaccination, with a notable exception. A sustained, multifunctional CD8(+) T-cell response against at least one intact class I epitope was detected in blood after vaccination. Persistence of HCV was not due to mutational escape of this epitope. Instead, failure to control HCV replication was likely caused by localized exhaustion in the liver, where CD8(+) T-cell expression of the inhibitory receptor programmed cell death 1 increased 25-fold compared with those in circulation. CONCLUSION: Treatment with a DAA during therapeutic vaccination provided transient control of HCV replication and a multifunctional T-cell response, primarily against nonconserved class I epitopes; exhaustion of liver-infiltrating CD8(+) T cells that target conserved epitopes may not be averted when DAA therapy fails prematurely due to emergence of resistant HCV variants.