Genetic Adaptation and Neandertal Admixture Shaped the Immune System of Human Populations.

Humans differ in the outcome that follows exposure to life-threatening pathogens, yet the extent of population differences in immune responses and their genetic and evolutionary determinants remain undefined. Here, we characterized, using RNA sequencing, the transcriptional response of primary monocytes from Africans and Europeans to bacterial and viral stimuli-ligands activating Toll-like receptor pathways (TLR1/2, TLR4, and TLR7/8) and influenza virus-and mapped expression quantitative trait loci (eQTLs). We identify numerous cis-eQTLs that contribute to the marked differences in immune responses detected within and between populations and a strong trans-eQTL hotspot at TLR1 that decreases expression of pro-inflammatory genes in Europeans only. We find that immune-responsive regulatory variants are enriched in population-specific signals of natural selection and show that admixture with Neandertals introduced regulatory variants into European genomes, affecting preferentially responses to viral challenges. Together, our study uncovers evolutionarily important determinants of differences in host immune responsiveness between human populations.

Dissecting human population variation in single-cell responses to SARS-CoV-2.

Humans display substantial interindividual clinical variability after SARS-CoV-2 infection1-3, the genetic and immunological basis of which has begun to be deciphered4. However, the extent and drivers of population differences in immune responses to SARS-CoV-2 remain unclear. Here we report single-cell RNA-sequencing data for peripheral blood mononuclear cells-from 222 healthy donors of diverse ancestries-that were stimulated with SARS-CoV-2 or influenza A virus. We show that SARS-CoV-2 induces weaker, but more heterogeneous, interferon-stimulated gene activity compared with influenza A virus, and a unique pro-inflammatory signature in myeloid cells. Transcriptional responses to viruses display marked population differences, primarily driven by changes in cell abundance including increased lymphoid differentiation associated with latent cytomegalovirus infection. Expression quantitative trait loci and mediation analyses reveal a broad effect of cell composition on population disparities in immune responses, with genetic variants exerting a strong effect on specific loci. Furthermore, we show that natural selection has increased population differences in immune responses, particularly for variants associated with SARS-CoV-2 response in East Asians, and document the cellular and molecular mechanisms by which Neanderthal introgression has altered immune functions, such as the response of myeloid cells to viruses. Finally, colocalization and transcriptome-wide association analyses reveal an overlap between the genetic basis of immune responses to SARS-CoV-2 and COVID-19 severity, providing insights into the factors contributing to current disparities in COVID-19 risk.

Human Immunity and Susceptibility to Influenza A(H3) Viruses of Avian, Equine, and Swine Origin.

Influenza A viruses (IAVs) of subtype H3 that infect humans are antigenically divergent from those of birds, horses, and swine. Human immunity against these viruses might be limited, implying potential pandemic risk. To determine human risk, we selected 4 avian, 1 equine, and 3 swine IAVs representing major H3 lineages. We tested serum collected during 2017-2018 from 286 persons in Belgium for hemagglutination inhibiting antibodies and virus neutralizing antibodies against those animal-origin IAVs and tested replication in human airway epithelia. Seroprevalence rates for circulating IAVs from swine in North America were >51%, swine in Europe 7%-37%, and birds and equids ≤12%. Replication was efficient for cluster IV-A IAVs from swine in North America and IAVs from swine in Europe, intermediate for IAVs from horses and poultry, and absent for IAVs from wild birds and a novel human-like swine IAV in North America. Public health risk may be highest for swine H3 IAVs.

A Randomized Dose-Escalating Phase I Trial of a Replication-Deficient Lymphocytic Choriomeningitis Virus Vector-Based Vaccine Against Human Cytomegalovirus.

BACKGROUND: A vaccine (HB-101) consisting of 2 nonreplicating lymphocytic choriomeningitis virus (LCMV) vectors expressing the human cytomegalovirus antigens glycoprotein B (gB) and the 65-kD phosphoprotein (pp65), respectively, is in development to prevent cytomegalovirus infection. METHODS: HB-101 was tested in cytomegalovirus-naive, healthy adults in a randomized, double-blind, placebo-controlled, dose-escalation Phase I trial. Fifty-four subjects received low, medium, or high dose of HB-101 or placebo by intramuscular administration at Month 0, 1, and 3. Safety and immunogenicity were the respective primary and secondary endpoints. Subjects were followed for 12 months after the initial immunization. RESULTS: Vaccination was associated with transient mild to moderate adverse events. HB-101 administration induced dose-dependent gB- and pp65-specific cellular responses, dominated by pp65-specific CD8 T cells, a high fraction of which were polyfunctional. Two administrations were sufficient to elicit dose-dependent gB-binding and cytomegalovirus-neutralizing antibodies (Abs). Cytomegalovirus-specific immune responses were boosted after each administration. Only 1 of 42 vaccine recipients mounted a transient LCMV vector-neutralizing Ab response. CONCLUSIONS: HB-101 was well tolerated and induced cytomegalovirus-specific polyfunctional CD8 T-cell and neutralizing Ab responses in the majority of subjects. Lack of vector-neutralizing Ab responses should facilitate booster vaccinations. These results justify further clinical evaluation of this vaccine candidate.

Safety and immunogenicity of two novel type 2 oral poliovirus vaccine candidates compared with a monovalent type 2 oral poliovirus vaccine in healthy adults: two clinical trials.

BACKGROUND: Two novel type 2 oral poliovirus vaccine (OPV2) candidates, novel OPV2-c1 and novel OPV2-c2, designed to be more genetically stable than the licensed Sabin monovalent OPV2, have been developed to respond to ongoing polio outbreaks due to circulating vaccine-derived type 2 polioviruses. METHODS: We did two randomised studies at two centres in Belgium. The first was a phase 4 historical control study of monovalent OPV2 in Antwerp, done before global withdrawal of OPV2, and the second was a phase 2 study in Antwerp and Ghent with novel OPV2-c1 and novel OPV2-c2. Eligible participants were healthy adults aged 18-50 years with documented history of at least three polio vaccinations, including OPV in the phase 4 study and either OPV or inactivated poliovirus vaccine (IPV) in the novel OPV2 phase 2 study, with no dose within 12 months of study start. In the historical control trial, participants were randomly assigned to either one dose or two doses of monovalent OPV2. In the novel OPV2 trial, participants with previous OPV vaccinations were randomly assigned to either one or two doses of novel OPV2-c1 or to one or two doses of novel OPV2-c2. IPV-vaccinated participants were randomly assigned to receive two doses of either novel OPV2-c1, novel OPV2-c2, or placebo. Vaccine administrators were unmasked to treatment; medical staff performing safety and reactogenicity assessments or blood draws for immunogenicity assessments were masked. Participants received the first vaccine dose on day 0, and a second dose on day 28 if assigned to receive a second dose. Primary objectives were assessments and comparisons of safety up to 28 days after each dose, including solicited adverse events and serious adverse events, and immunogenicity (seroprotection rates on day 28 after the first vaccine dose) between monovalent OPV2 and the two novel OPV2 candidates. Primary immunogenicity analyses were done in the per-protocol population. Safety was assessed in the total vaccinated population-ie, all participants who received at least one dose of their assigned vaccine. The phase 4 control study is registered with EudraCT (2015-003325-33) and the phase 2 novel OPV2 study is registered with EudraCT (2018-001684-22) and ClinicalTrials.gov (NCT04544787). FINDINGS: In the historical control study, between Jan 25 and March 18, 2016, 100 volunteers were enrolled and randomly assigned to receive one or two doses of monovalent OPV2 (n=50 in each group). In the novel OPV2 study, between Oct 15, 2018, and Feb 27, 2019, 200 previously OPV-vaccinated volunteers were assigned to the four groups to receive one or two doses of novel OPV2-c1 or novel OPV2-c2 (n=50 per group); a further 50 participants, previously vaccinated with IPV, were assigned to novel OPV2-c1 (n=17), novel OPV2-c2 (n=16), or placebo (n=17). All participants received the first dose of assigned vaccine or placebo and were included in the total vaccinated population. All vaccines appeared safe; no definitely vaccine-related withdrawals or serious adverse events were reported. After first doses in previously OPV-vaccinated participants, 62 (62%) of 100 monovalent OPV2 recipients, 71 (71%) of 100 recipients of novel OPV2-c1, and 74 (74%) of 100 recipients of novel OPV2-c2 reported solicited systemic adverse events, four (monovalent OPV2), three (novel OPV2-c1), and two (novel OPV2-c2) of which were considered severe. In IPV-vaccinated participants, solicited adverse events occurred in 16 (94%) of 17 who received novel OPV2-c1 (including one severe) and 13 (81%) of 16 who received novel OPV2-c2 (including one severe), compared with 15 (88%) of 17 placebo recipients (including two severe). In previously OPV-vaccinated participants, 286 (97%) of 296 were seropositive at baseline; after one dose, 100% of novel OPV2 vaccinees and 97 (97%) of monovalent OPV2 vaccinees were seropositive. INTERPRETATION: Novel OPV2 candidates were as safe, well tolerated, and immunogenic as monovalent OPV2 in previously OPV-vaccinated and IPV-vaccinated adults. These data supported the further assessment of the vaccine candidates in children and infants. FUNDING: University of Antwerp and Bill & Melinda Gates Foundation.

Safety and Immunogenicity of a Second Dose of an Investigational Maternal Trivalent Group B Streptococcus Vaccine in Nonpregnant Women 4-6 Years After a First Dose: Results From a Phase 2 Trial.

BACKGROUND: Maternal immunization against group B streptococcus (GBS) could protect infants from invasive GBS disease. Additional doses in subsequent pregnancies may be needed. We evaluated the safety and immunogenicity of a second dose of an investigational trivalent CRM197-glycoconjugate GBS vaccine (targeting serotypes Ia/Ib/III), administered to nonpregnant women 4-6 years postdose 1. METHODS: Healthy women either previously vaccinated with 1 dose of trivalent GBS vaccine 4-6 years before enrollment (n = 53) or never GBS vaccinated (n = 27) received a single trivalent GBS vaccine injection. Adverse events (AEs) were recorded. Serotype-specific (Ia/Ib/III) anti-GBS antibodies were measured by multiplex immunoassay prevaccination and 30/60 days postvaccination. RESULTS: AEs were reported with similar rates after a first or second dose; none were serious. Of previously GBS-vaccinated women, 92%-98% had anti-GBS concentrations that exceeded an arbitrary threshold (8 µg/mL) for each serotype 60 days postdose 2 vs 36%-56% postdose 1 in previously non-GBS-vaccinated women. Of previously GBS-vaccinated women with undetectable baseline (predose 1) anti-GBS levels, 90%-98% reached this threshold postdose 2. For each serotype, anti-GBS geometric mean concentrations (GMCs) 30/60 days postdose 2 in previously GBS-vaccinated women were ≥200-fold higher than baseline GMCs. Among women with undetectable baseline anti-GBS levels, postdose 2 GMCs in previously GBS-vaccinated women exceeded postdose 1 GMCs in previously non-GBS-vaccinated women (≥7-fold). CONCLUSIONS: A second trivalent GBS vaccine dose administered 4-6 years postdose 1 was immunogenic with a favorable safety profile. Women with undetectable preexisting anti-GBS concentrations may benefit from a sufficiently spaced second vaccine dose. CLINICAL TRIALS REGISTRATION: NCT02690181.

Detection of H1 Swine Influenza A Virus Antibodies in Human Serum Samples by Age Group1.

Most H1 influenza A viruses (IAVs) of swine are derived from past human viruses. As human population immunity against these IAVs gradually decreases, the risk of reintroduction to humans increases. We examined 549 serum samples from persons 0-97 years of age collected in Belgium during 2017-2018 for hemagglutination inhibiting and virus neutralizing antibodies against 7 major H1 swine IAV (swIAV) clades and 3 human progenitor IAVs. Seroprevalence (titers >40) rates were >50% for classical swine and European human-like swIAVs, >24% for North American human-like δ1a and Asian avian-like swIAVs, and <10% for North American human-like δ1b and European avian-like swIAVs, but rates were age-dependent. Antibody titers against human-like swIAVs and supposed human precursor IAVs correlated with correlation coefficients of 0.30-0.86. Our serologic findings suggest that European avian-like, clade 1C.2.1, and North American human-like δ1b, clade 1B.2.2.2, H1 swIAVs pose the highest pandemic risk.

Immunogenicity and Safety of 3 Formulations of a Respiratory Syncytial Virus Candidate Vaccine in Nonpregnant Women: A Phase 2, Randomized Trial.

BACKGROUND: Respiratory syncytial virus (RSV) is a common cause of respiratory tract illness and hospitalization in neonates and infants. RSV vaccination during pregnancy may protect offspring in their first months of life. METHODS: This randomized, observer-blind, multicenter, phase 2 study evaluated the immunogenicity and safety of an RSV candidate vaccine in healthy nonpregnant women aged 18-45 years. Four hundred participants were randomized (1:1:1:1) to receive a single intramuscular dose of vaccine containing 30 µg, 60 µg, or 120 µg of RSV fusion protein engineered to preferentially maintain a prefusion conformation (RSV-PreF vaccine) or placebo. RESULTS: Thirty days postvaccination, RSV-A neutralizing antibody geometric mean titers (GMTs) increased 3.75-, 4.42- and 4.36-fold; RSV-B neutralizing antibody GMTs 2.36-, 2.54- and 2.76-fold; and palivizumab competing antibody (PCA) concentrations 11.69-, 14.38- and 14.24-fold compared with baseline levels in the 30 µg, 60 µg, and 120 µg RSV-PreF groups, respectively. Antibody titers and PCA concentrations at day 30 were significantly higher with the 120 µg compared to the 30 µg RSV-PreF vaccine. All RSV-PreF vaccine formulations and the placebo had similar reactogenicity profiles. No serious adverse events were considered to be related to the RSV-PreF vaccine. CONCLUSIONS: The 3 formulations of the investigational RSV-PreF vaccine were well-tolerated and induced RSV-A and RSV-B neutralizing antibodies and PCAs in healthy, nonpregnant women. CLINICAL TRIALS REGISTRATION: NCT02956837.

Persistence of HBsAg-specific antibodies and immune memory two to three decades after hepatitis B vaccination in adults.

The duration of protection after hepatitis B vaccination is not exactly known. This phase IV study evaluated antibody persistence and immune memory 20-30 years after adult immunization with recombinant hepatitis B vaccine (HBsAg vaccine, Engerix-B) in routine clinical practice. Men and women 40-60 years old, with documented evidence of vaccination with three or four HBsAg vaccine doses 20-30 years earlier and without subsequent booster, were enrolled and received HBsAg vaccine as challenge dose. HBsAg-specific antibodies (anti-HBs) and frequencies of HBsAg-specific circulating memory B cells and CD4+ T cells expressing combinations of activation markers (CD40L, IL2, IFNγ, TNFα) were measured prechallenge, 7 and 30 days postchallenge. Of 101 participants in the according-to-protocol cohort for immunogenicity, 90.1% had anti-HBs concentrations ≥ 10 mIU/mL prechallenge administration; 84.2% and 100% mounted an anamnestic response 7 and 30 days postchallenge, respectively. HBsAg-specific memory B and CD4+ T cells expressing at least two activation markers were low prechallenge and increased markedly postchallenge. These results suggest sustained immune memory and long-term protection 20-30 years after a complete primary HBsAg vaccination course during adulthood, in line with current recommendations that a booster is not needed in fully vaccinated immunocompetent adults.

Safety and Immunogenicity of Different Formulations of Norovirus Vaccine Candidate in Healthy Adults: A Randomized, Controlled, Double-Blind Clinical Trial.

Background: We investigated safety and immunogenicity of 1-2 doses of different bivalent virus-like particle (VLP) norovirus vaccine candidate (NoV) formulations in healthy 18- to 64-year-olds. Methods: On days 1 and 28, participants (n = 420) randomized to 14 equal groups received intramuscular control vaccine (hepatitis A) or 1 of 11 NoV formulations containing varying dosages of GI.1 and GII.4c genotype VLP antigens with aluminum hydroxide [Al(OH)3], and 0 µg, 15 µg, or 50 µg monophosphoryl lipid A (MPL). Immunogenicity was assessed on days 1, 28, 56, 208 and 393. Solicited local and systemic reactions were recorded for 7 days, unsolicited adverse events (AEs) until day 56, and serious AEs throughout the trial. Results: All NoV formulations induced similar increases in pan-immunoglobulin, immunoglobulin A, and histo-blood group binding antigen-blocking antibodies by day 56, mostly after 1 dose, that persisted above baseline to day 393. Higher GI.1 content interfered with GII.4c responses, and responses did not benefit from MPL. Overall reactogenicity consisted of mainly mild injection site pain, headache, and fatigue. No vaccine-related serious AEs were reported. Conclusions: All candidate NoV formulations were well tolerated. Overall, 15 µg GI.1/50 µg GII.4c elicited the best balance of immunogenicity with no clear benefit of MPL, and is the candidate formulation being taken forward in clinical development. Clinical Trials Registration: NCT02038907.

Study of hepatitis E virus infection of genotype 1 and 3 in mice with humanised liver.

OBJECTIVE: The hepatitis E virus (HEV) is responsible for approximately 20 million infections per year worldwide. Although most infected people can spontaneously clear an HEV infection, immune-compromised individuals may evolve towards chronicity. Chronic HEV infection can be cured using ribavirin, but viral isolates with low ribavirin sensitivity have recently been identified. Although some HEV isolates can be cultured in vitro, in vivo studies are essentially limited to primates and pigs. Since the use of these animals is hampered by financial, practical and/or ethical concerns, we evaluated if human liver chimeric mice could serve as an alternative. DESIGN: Humanised mice were inoculated with different HEV-containing preparations. RESULTS: Chronic HEV infection was observed after intrasplenic injection of cell culture-derived HEV, a filtered chimpanzee stool suspension and a patient-derived stool suspension. The viral load was significantly higher in the stool compared with the plasma. Overall, the viral titre in genotype 3-infected mice was lower than that in genotype 1-infected mice. Analysis of liver tissue of infected mice showed the presence of viral RNA and protein, and alterations in host gene expression. Intrasplenic injection of HEV-positive patient plasma and oral inoculation of filtered stool suspensions did not result in robust infection. Finally, we validated our model for the evaluation of novel antiviral compounds against HEV using ribavirin. CONCLUSIONS: Human liver chimeric mice can be infected with HEV of different genotypes. This small animal model will be a valuable tool for the in vivo study of HEV infection and the evaluation of novel antiviral molecules.

Monoclonal anti-envelope antibody AP33 protects humanized mice against a patient-derived hepatitis C virus challenge.

UNLABELLED: End-stage liver disease (ESLD) caused by hepatitis C virus (HCV) infection is a major indication for liver transplantation. However, immediately after transplantation, the liver graft of viremic patients universally becomes infected by circulating virus, resulting in accelerated liver disease progression. Currently available direct-acting antiviral therapies have reduced efficacy in patients with ESLD and prophylactic strategies to prevent HCV recurrence are still highly needed. In this study, we compared the ability of two broadly reactive monoclonal antibodies (mAbs), designated 3/11 and AP33, recognizing a distinct, but overlapping, epitope in the viral E2 glycoprotein to protect humanized mice from a patient-derived HCV challenge. Their neutralizing activity was assessed using the HCV pseudoparticles and cell-culture-derived HCV systems expressing multiple patient-derived envelopes and a human-liver chimeric mouse model. HCV RNA was readily detected in all control mice challenged with a patient-derived HCV genotype 1b isolate, whereas 3 of 4 AP33-treated mice were completely protected. In contrast, only one of four 3/11-treated mice remained HCV-RNA negative throughout the observation period, whereas the other 3 had a viral load that was indistinguishable from that in the control group. The increased in vivo efficacy of AP33 was in line with its higher affinity and neutralizing capacity observed in vitro. CONCLUSIONS: Although mAbs AP33 and 3/11 target the same region in E2, only mAb AP33 can efficiently protect from challenge with a heterologous HCV population in vivo. Given that mAb AP33 efficiently neutralizes viral variants that escaped the humoral immune response and reinfected the liver graft of transplant patients, it may be a valuable candidate to prevent HCV recurrence. In addition, our data are valuable for the design of a prophylactic vaccine.

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.

Generation and characterization of small single domain antibodies inhibiting human tumor necrosis factor receptor 1.

The cytokine TNF is a well known drug target for several inflammatory diseases such as Crohn disease. Despite the great success of TNF blockers, therapy could be improved because of high costs and side effects. Selective inhibition of TNF receptor (TNFR) 1 signaling holds the potential to greatly reduce the pro-inflammatory activity of TNF, thereby preserving the advantageous immunomodulatory signals mediated by TNFR2. We generated a selective human TNFR1 inhibitor based on Nanobody (Nb) technology. Two anti-human TNFR1 Nbs were linked with an anti-albumin Nb to generate Nb Alb-70-96 named "TNF Receptor-One Silencer" (TROS). TROS selectively binds and inhibits TNF/TNFR1 and lymphotoxin-α/TNFR1 signaling with good affinity and IC50 values, both of which are in the nanomolar range. Surface plasmon resonance analysis reveals that TROS competes with TNF for binding to human TNFR1. In HEK293T cells, TROS strongly reduces TNF-induced gene expression, like IL8 and TNF, in a dose-dependent manner; and in ex vivo cultured colon biopsies of CD patients, TROS inhibits inflammation. Finally, in liver chimeric humanized mice, TROS antagonizes inflammation in a model of acute TNF-induced liver inflammation, reflected in reduced human IL8 expression in liver and reduced IL6 levels in serum. These results demonstrate the considerable potential of TROS and justify the evaluation of TROS in relevant disease animal models of both acute and chronic inflammation and eventually in patients.

Impact of adjuvants on CD4(+) T cell and B cell responses to a protein antigen vaccine: Results from a phase II, randomized, multicenter trial.

Immunogenicity and safety of different adjuvants combined with a model antigen (HBsAg) were compared. Healthy HBV-naïve adults were randomized to receive HBs adjuvanted with alum or Adjuvant Systems AS01B, AS01E, AS03A or AS04 at Days 0 and 30. Different frequencies of HBs-specific CD4+ T cells 14days post dose 2 but similar polyfunctionality profiles were induced by the different adjuvants with frequencies significantly higher in the AS01B and AS01E groups than in the other groups. Antibody concentrations 30days post-dose 2 were significantly higher in AS01B, AS01E and AS03A than in other groups. Limited correlations were observed between HBs-specific CD4+ T cell and antibody responses. Injection site pain was the most common solicited local symptom and was more frequent in AS groups than in alum group. Different adjuvants formulated with the same antigen induced different adaptive immune responses and reactogenicity patterns in healthy naïve adults. The results summary for this study (GSK study number 112115 - NCT# NCT00805389) is available on the GSK Clinical Study Register and can be accessed at www.gsk-clinicalstudyregister.com.

A monoclonal antibody-based immunoassay to measure the antibody response against the repeat region of the circumsporozoite protein of Plasmodium falciparum.

BACKGROUND: The malaria vaccine candidate RTS,S/AS01 (GSK Vaccines) induces high IgG concentration against the circumsporozoite protein (CSP) of Plasmodium falciparum. In human vaccine recipients circulating anti-CSP antibody concentrations are associated with protection against infection but appear not to be the correlate of protection. However, in a humanized mouse model of malaria infection prophylactic administration of a human monoclonal antibody (MAL1C), derived from a RTS,S/AS01-immunized volunteer, directed against the CSP repeat region, conveyed full protection in a dose-dependent manner suggesting that antibodies alone are able to prevent P. falciparum infection when present in sufficiently high concentrations. A competition ELISA was developed to measure the presence of MAL1C-like antibodies in polyclonal sera from RTS,S/AS01 vaccine recipients and study their possible contribution to protection against infection. RESULTS: MAL1C-like antibodies present in polyclonal vaccine-induced sera were evaluated for their ability to compete with biotinylated monoclonal antibody MAL1C for binding sites on the capture antigen consisting of the recombinant protein encompassing 32 NANP repeats of CSP (R32LR). Serum samples were taken at different time points from participants in two RTS,S/AS01 vaccine studies (NCT01366534 and NCT01857869). Vaccine-induced protection status of the study participants was determined based on the outcome of experimental challenge with infected mosquito bites after vaccination. Optimal conditions were established to reliably detect MAL1C-like antibodies in polyclonal sera. Polyclonal anti-CSP antibodies and MAL1C-like antibody content were measured in 276 serum samples from RTS,S/AS01 vaccine recipients using the standard ELISA and MAL-1C competition ELISA, respectively. A strong correlation was observed between the results from these assays. However, no correlation was found between the results of either assay and protection against infection. CONCLUSIONS: The competition ELISA to measure MAL1C-like antibodies in polyclonal sera from RTS,S/AS01 vaccine recipients was robust and reliable but did not reveal the elusive correlate of protection.

In vitro and in vivo metabolism studies of dimethazine.

The use of anabolic steroids is prohibited in sports. Effective control is done by monitoring their metabolites in urine samples collected from athletes. Ethical objections however restrict the use of designer steroids in human administration studies. To overcome these problems alternative in vitro and in vivo models were developed to identify metabolites and to assure a fast response by anti-doping laboratories to evolutions on the steroid market. In this study human liver microsomes and an uPA(+/+) -SCID chimeric mouse model were used to elucidate the metabolism of a steroid product called 'Xtreme DMZ'. This product contains the designer steroid dimethazine (DMZ), which consists of two methasterone molecules linked by an azine group. In the performed stability study, degradation from dimethazine to methasterone was observed. By a combination of LC-High Resolution Mass Spectrometry (HRMS) and GC-MS(/MS) analysis methasterone and six other dimethazine metabolites (M1-M6), which are all methasterone metabolites, could be detected besides the parent compound in both models. The phase II metabolism of dimethazine was also investigated in the mouse urine samples. Only metabolites M1 and M2 were exclusively detected in the glucuro-conjugated fraction; all other compounds were also found in the free fraction. For effective control of DMZ misuse in doping control samples, screening for methasterone and methasterone metabolites should be sufficient. Copyright © 2016 John Wiley & Sons, Ltd.

Anti-CD81 but not anti-SR-BI blocks Plasmodium falciparum liver infection in a humanized mouse model.

OBJECTIVES: Plasmodium falciparum sporozoites, deposited in the skin by infected Anopheles mosquitoes taking a blood meal, cross the endothelium of skin capillaries and travel to the liver where they traverse Kupffer cells and hepatocytes to finally invade a small number of the latter. In hepatocytes, sporozoites replicate, differentiate and give rise to large numbers of merozoites that are released into the bloodstream where they invade red blood cells, thus initiating the symptomatic blood stage. Using in vitro systems and rodent models, it has been shown that the hepatocyte receptors CD81 and scavenger receptor type B class I (SR-BI) play a pivotal role during sporozoite invasion. We wanted to evaluate whether these two entry factors are genuine drug targets for the prevention of P. falciparum infection in humans. METHODS: Immunodeficient mice of which the liver is largely repopulated by human hepatocytes were treated with monoclonal antibodies blocking either CD81 or SR-BI 1 day prior to challenge with infected mosquitoes. P. falciparum infection of the liver was demonstrated using a qPCR assay. RESULTS: In human liver chimeric mice, an antibody directed against CD81 completely blocked P. falciparum sporozoite invasion while SR-BI-specific monoclonal antibodies did not influence in vivo infection. CONCLUSIONS: These observations confirm the role of CD81 in liver-stage malaria and question that of SR-BI. CD81 might be a valuable drug target for the prevention of malaria.

Combined adenovirus vector and hepatitis C virus envelope protein prime-boost regimen elicits T cell and neutralizing antibody immune responses.

UNLABELLED: Despite the recent progress in the development of new antiviral agents, hepatitis C virus (HCV) infection remains a major global health problem, and there is a need for a preventive vaccine. We previously reported that adenoviral vectors expressing HCV nonstructural proteins elicit protective T cell responses in chimpanzees and were immunogenic in healthy volunteers. Furthermore, recombinant HCV E1E2 protein formulated with adjuvant MF59 induced protective antibody responses in chimpanzees and was immunogenic in humans. To develop an HCV vaccine capable of inducing both T cell and antibody responses, we constructed adenoviral vectors expressing full-length and truncated E1E2 envelope glycoproteins from HCV genotype 1b. Heterologous prime-boost immunization regimens with adenovirus and recombinant E1E2 glycoprotein (genotype 1a) plus MF59 were evaluated in mice and guinea pigs. Adenovirus prime and protein boost induced broad HCV-specific CD8+ and CD4+ T cell responses and functional Th1-type IgG responses. Immune sera neutralized luciferase reporter pseudoparticles expressing HCV envelope glycoproteins (HCVpp) and a diverse panel of recombinant cell culture-derived HCV (HCVcc) strains and limited cell-to-cell HCV transmission. This study demonstrated that combining adenovirus vector with protein antigen can induce strong antibody and T cell responses that surpass immune responses achieved by either vaccine alone. IMPORTANCE: HCV infection is a major health problem. Despite the availability of new directly acting antiviral agents for treating chronic infection, an affordable preventive vaccine provides the best long-term goal for controlling the global epidemic. This report describes a new anti-HCV vaccine targeting the envelope viral proteins based on adenovirus vector and protein in adjuvant. Rodents primed with the adenovirus vaccine and boosted with the adjuvanted protein developed cross-neutralizing antibodies and potent T cell responses that surpassed immune responses achieved with either vaccine component alone. If combined with the adenovirus vaccine targeting the HCV NS antigens now under clinical testing, this new vaccine might lead to a stronger and broader immune response and to a more effective vaccine to prevent HCV infection. Importantly, the described approach represents a valuable strategy for other infectious diseases in which both T and B cell responses are essential for protection.

Successful anti-scavenger receptor class B type I (SR-BI) monoclonal antibody therapy in humanized mice after challenge with HCV variants with in vitro resistance to SR-BI-targeting agents.

UNLABELLED: Hepatitis C virus (HCV)-induced endstage liver disease is currently a major indication for liver transplantation. After transplantation the donor liver inevitably becomes infected with the circulating virus. Monoclonal antibodies (mAbs) against the HCV coreceptor scavenger receptor class B type I (SR-BI) inhibit HCV infection of different genotypes, both in cell culture and in humanized mice. Anti-SR-BI mAb therapy is successful even when initiated several days after HCV exposure, supporting its potential applicability to prevent HCV reinfection of liver allografts. However, HCV variants with reduced SR-BI dependency have been described in the literature, which could potentially limit the use of SR-BI targeting therapy. In this study we show, both in a preventative and postexposure setting, that humanized mice infected with HCV variants exhibiting increased in vitro resistance to SR-BI-targeting molecules remain responsive to anti-SR-BI mAb therapy in vivo. A 2-week antibody therapy readily cleared HCV RNA from the circulation of infected humanized mice. We found no evidence supporting increased SR-BI-receptor dependency of viral particles isolated from humanized mice compared to cell culture-produced virus. However, we observed that, unlike wild-type virus, the in vitro infectivity of the resistant variants was inhibited by both human high density lipoprotein (HDL) and very low density lipoprotein (VLDL). The combination of mAb1671 with these lipoproteins further increased the antiviral effect. CONCLUSION: HCV variants that are less dependent on SR-BI in vitro can still be efficiently blocked by an anti-SR-BI mAb in humanized mice. Since these variants are also more susceptible to neutralization by anti-HCV envelope antibodies, their chance of emerging during anti-SR-BI therapy is severely reduced. Our data indicate that anti-SR-BI receptor therapy could be an effective way to prevent HCV infection in a liver transplant setting.