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Seasonal influenza vaccine effectiveness is low. Carbohydrate fatty acid monosulphate ester (CMS), a new oil-in-water adjuvant, has proven potency in animal models with suggested capacity for dose-sparing. The objective was to evaluate safety and immunogenicity of CMS when added to a low-dose influenza vaccine (QIV) in humans. In a randomised, double-blind, active-controlled, first-in-human study, sixty participants (18–50 years) received either 0.5 mg CMS or 2 mg CMS with 1/5th dose QIV, or a full dose QIV without CMS. Adverse events (AE) were monitored until 7 days post-vaccination. Haemagglutinin inhibition (HI) titres in serum and CD4+ T cells in PBMCs were determined at day 0, 7, 28, and 180. Mean age was 37.6 (±10.1) years and 42/60 (70.0%) were female. Pain at injection site (42/60, 86.7%) and headache (34/60, 56.7%) were reported most and more frequently in the 2 mg CMS group. HI titres and the frequency of influenza specific CD4+ T cells were equal across strains for the three cohorts on all visits, increased until day 28 and decreased at day 180 to values higher than baseline. CMS was safe in humans. Humoral and cell-mediated immunogenicity was similar across vaccines, even with 1/5th antigen dose. CMS can have beneficial implications in low-resource settings or in a pandemic context.

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.

The development of an efficacious vaccine against norovirus is of paramount importance given its potential to reduce the global burden of norovirus-associated morbidity and mortality. Here, we report a detailed immunological analysis of a phase I, double-blind, placebo-controlled clinical trial performed on 60 healthy adults, ages 18 to 40. Total serum immunoglobulin and serum IgA against vaccine strains and cross-reactive serum IgG against non-vaccine strains were measured by enzyme immunoassays, whereas cell-mediated immune responses were quantified using intracellular cytokine staining by flow cytometry. A significant increase in humoral and cellular responses, e.g., IgA and CD4+ polypositive T cells, was triggered by the GI.4 Chiba 407 (1987) and GII.4 Aomori 2 (2006) VLP-based norovirus vaccine candidate rNV-2v, which is formulated without adjuvant. No booster effect was observed after the second administration in the pre-exposed adult study population. Furthermore, a cross-reactive immune response was elicited, as shown by IgG titers against GI.3 (2002), GII.2 OC08154 (2008), GII.4 (1999), GII.4 Sydney (2012), GII.4 Washington (2018), GII.6 Maryland (2018), and GII.17 Kawasaki 308 (2015). Due to viral infection via mucosal gut tissue and the high variety of potentially relevant norovirus strains, a focus should be on IgA and cross-protective humoral and cell-mediated responses in the development of a broadly protective, multi-valent norovirus vaccine.Clinical trial registrationhttps://clinicaltrials.gov, identifier NCT05508178. EudraCT number: 2019-003226-25.

Background & Aims: Elimination of chronic HBV/HDV infection remains a major global health challenge. Targeting excessive hepatitis B surface antigen (HBsAg) release may provide an interesting window of opportunity to break immune tolerance and to achieve a functional cure using additional antivirals. Methods: We evaluated a HBsAg-specific human monoclonal antibody, as part of either a prophylactic or therapeutic strategy, against HBV/HDV infection in cell culture models and in human-liver chimeric mice. To assess prophylactic efficacy, mice were passively immunized prior to infection with HBV or HBV/HDV (coinfection and superinfection setting). Therapeutic efficacy was assessed in HBV and HBV/HDV-coinfected mice receiving 4 weeks of treatment. Viral parameters (HBV DNA, HDV RNA and HBsAg) were assessed in mouse plasma. Results: The antibody could effectively prevent HBV/HDV infection in a dose-dependent manner with IC50 values of ∼3.5 ng/ml. Passive immunization showed complete protection of mice from both HBV and HBV/HDV coinfection. Moreover, HDV superinfection was either completely prevented or at least attenuated in HBV-infected mice. Finally, antibody treatment in mice with established HBV/HDV infection resulted in a significant decline in viremia and a concomitant drop in on-treatment HBsAg, with a moderate viral rebound following treatment cessation. Conclusion: We present data on a valuable antibody candidate that could complement other antivirals in strategies aimed at achieving functional cure of chronic HBV and HDV infection. Impact and implications: Patients chronically infected with HBV may eventually develop liver cancer and are at great risk of being superinfected with HDV, which worsens and accelerates disease progression. Unfortunately, current treatments can rarely eliminate both viruses from chronically infected patients. In this study, we present data on a novel antibody that is able to prevent chronic HBV/HDV infection in a mouse model with a humanized liver. Moreover, antibody treatment of HBV/HDV-infected mice strongly diminishes viral loads during therapy. This antibody is a valuable candidate for further clinical development.

A single birth-dose of Hepatitis B vaccine (HepB) can protect newborns from acquiring Hepatitis B infection through vertical transmission, though several follow-up doses are required to induce long-lived protection. In addition to stimulating antibodies, a birth-dose of HepB might also induce polyfunctional CD4+ T-cells, which may contribute to initial protection. We investigated whether vaccination with HepB in the first week of life induced detectable antigen-specific CD4+ T-cells after only a single dose and following completion of the entire HepB vaccine schedule (3 doses). Using HBsAg- stimulated peripheral blood mononuclear cells from 344 infants, we detected increased populations of antigen-specific polyfunctional CD154+IL-2+TNFα+ CD4+ T-cells following a single birth-dose of HepB in a proportion of infants. Frequencies of polyfunctional T-cells increased following the completion of the HepB schedule but increases in the proportion of responders as compared to following only one dose was marginal. Polyfunctional T-cells correlated positively with serum antibody titres following the birth dose (day30) and completion of the 3-dose primary HepB vaccine series (day 128). These data indicate that a single birth dose of HepB provides immune priming for both antigen-specific B- and T cells

Noroviruses (NoV) are the leading cause of epidemic acute gastroenteritis in humans worldwide and a safe and effective vaccine is needed. Here, a phase I, double-blind, placebo-controlled clinical trial was performed in 60 healthy adults, 18 to 40 years old. Safety (primary objective) and immunogenicity (secondary and exploratory objectives) of a bivalent (GI.4 and GII.4), plant-produced, virus-like particle (VLP), NoV vaccine candidate formulation were investigated at two dose levels (50 µg + 50 µg and 150 µg + 150 µg) without adjuvant. Overall, 13 subjects (65.0%) in the 50 µg group, 16 subjects (80.0%) in the 150 µg group, and 14 subjects (70.0%) in the placebo group reported at least 1 solicited local or general symptom during the 7-day post-vaccination periods following each dose. Severe solicited adverse events (AEs) were rare (2 events in the 50 µg group). A total of 8 subjects (40.0%) in each group reported at least one unsolicited AE during the 28-day post-vaccination periods. Immunogenicity was assessed on days 1, 8, 29, 57, 183 and 365. All subjects were pre-exposed to norovirus as indicated by baseline levels of the different immunological parameters examined. Vaccine-specific humoral and cellular immune responses increased after the first dose but did not rise further after the second vaccination. Increased GI.4- and GII.4-specific IgG titers persisted until day 365. The vaccine elicited cross-reactive IgG antibodies against non-vaccine NoV VLPs, which was more pronounced for NoV strains of the same genotype as the GII.4 vaccine strain than for non-vaccine genotypes. Significant blocking anti-GI.4 and anti-GII.4 VLP titers were triggered in both dose groups. Lymphoproliferation assays revealed strong cell-mediated immune responses that persisted until day 365. In conclusion, both dose levels were safe and well-tolerated, and no higher incidence of AEs was observed in the higher dose group. The data show that a single dose of the vaccine formulated at 50 µg of each VLP is sufficient to reach a peak immune response after 8 to 28 days. The results of this Phase I study warrant further evaluation of the non-adjuvanted vaccine candidate.Clinical trial registrationhttps://clinicaltrials.gov/ct2/show/record/NCT05508178, identifier (NCT05508178).

Despite the knowledge that cell-mediated immunity (CMI) contributes to the reduction of severe influenza infection, transmission, and disease outcome, the correlates of protection for cell-mediated immunity remain still unclear. Therefore, measuring the magnitude and quality of influenza-specific T cell responses in a harmonized way is of utmost importance to improve characterisation of vaccine-induced immunity across different clinical trials. The present study, conducted as part of the FLUCOP project, describes the development of a consensus protocol for the intracellular cytokine staining (ICS) assay, in order to reduce inter-laboratory variability, and its qualification. In order to develop a consensus protocol, the study was divided into different stages. Firstly, two pilot studies evaluated critical parameters in the analytical (read-outs) and post-analytical (gating strategies and data analysis) methods applied by eight different laboratories within the FLUCOP consortium. The methods were then harmonized by fixing the critical parameters and the subsequent consensus protocol was then qualified by one FLUCOP member. The antigen-specific cell population was defined as polypositive CD4+ T cells (i.e. positive for at least two markers among CD40L/IFNγ/IL2/TNFα), which was shown to be the most sensitive and specific read-out. The qualification of this consensus protocol showed that the quantification of polypositive CD4+ T cells was precise, linear and accurate, and sensitive with a lower limit of quantification of 0.0335% antigen-specific polypositive CD4+ T cells. In conclusion, we provide the description of a harmonized ICS assay, which permits quantitative and qualitative evaluation of influenza vaccine-induced T cell responses. Application of this harmonized assay may allow for future comparisons of T cell responses to different influenza vaccines. It may facilitate future assessments of potential correlates of protection with the promise of application across other pathogens.

Influenza continues to be the most important cause of viral respiratory disease, despite the availability of vaccines. Today’s evaluation of influenza vaccines mainly focuses on the quantitative and functional analyses of antibodies to the surface proteins haemagglutinin (HA) and neuraminidase (NA). However, there is an increasing interest in measuring cellular immune responses targeting not only mutation-prone surface HA and NA but also conserved internal proteins as these are less explored yet potential correlates of protection. To date, laboratories that monitor cellular immune responses use a variety of in-house procedures. This generates diverging results, complicates interlaboratory comparisons, and hampers influenza vaccine evaluation. The European FLUCOP project aims to develop and standardize assays for the assessment of influenza vaccine correlates of protection. This report describes the harmonization and qualification of the influenza-specific interferon-gamma (IFN-γ) Enzyme-Linked ImmunoSpot (ELISpot) assay. Initially, two pilot studies were conducted to identify sources of variability during sample analysis and spot enumeration in order to develop a harmonized Standard Operating Procedure (SOP). Subsequently, an assay qualification study was performed to investigate the linearity, intermediate precision (reproducibility), repeatability, specificity, Lower and Upper Limits of Quantification (LLOQ-ULOQ), Limit of Detection (LOD) and the stability of signal over time. We were able to demonstrate that the FLUCOP harmonized IFN-γ ELISpot assay procedure can accurately enumerate IFN-γ secreting cells in the analytical range of 34.4 Spot Forming Units (SFU) per million cells up to the technical limit of the used reader and in the linear range from 120 000 to 360 000 cells per well, in plates stored up to 6 weeks after development. This IFN-γ ELISpot procedure will hopefully become a useful and reliable tool to investigate influenza-specific cellular immune responses induced by natural infection or vaccination and can be an additional instrument in the search for novel correlates of protection.

Influenza vaccines remain the most effective tools to prevent flu and its complications. Trivalent or quadrivalent inactivated influenza vaccines primarily elicit antibodies towards haemagglutinin and neuraminidase. These vaccines fail to induce high protective efficacy, in particular in older adults and immunocompromised individuals and require annual updates to keep up with evolving influenza strains (antigenic drift). Vaccine efficacy declines when there is a mismatch between its content and circulating strains. Current correlates of protection are merely based on serological parameters determined by haemagglutination inhibition or single radial haemolysis assays. However, there is ample evidence showing that these serological correlates of protection can both over- or underestimate the protective efficacy of influenza vaccines. Next-generation universal influenza vaccines that induce cross-reactive cellular immune responses (CD4+ and/or CD8+ T-cell responses) against conserved epitopes may overcome some of the shortcomings of the current inactivated vaccines by eliciting broader protection that lasts for several influenza seasons and potentially enhances pandemic preparedness. Assessment of cellular immune responses in clinical trials that evaluate the immunogenicity of these new generation vaccines is thus of utmost importance. Moreover, studies are needed to examine whether these cross-reactive cellular immune responses can be considered as new or complementary correlates of protection in the evaluation of traditional and next-generation influenza vaccines. An overview of the assays that can be applied to measure cell-mediated immune responses to influenza with their strengths and weaknesses is provided here.

Background: The COVID-19 vaccine candidate CVnCoV comprises sequence-optimized mRNA encoding SARS-CoV-2 S-protein encapsulated in lipid nanoparticles. In this phase 2a study, we assessed reactogenicity and immunogenicity of two or three doses in younger and older adults. Methods: Younger (18–60 years) and older (>60 years) adults were enrolled in two sites in Panama and Peru to receive either 6 or 12 µg doses of CVnCoV or licensed control vaccines 28 days apart; subsets received a 12 µg booster dose on Day 57 or Day 180. Solicited adverse events (AE) were reported for 7 days and unsolicited AEs for 4 weeks after each vaccination, and serious AEs (SAE) throughout the study. Humoral immunogenicity was measured as neutralizing and receptor binding domain (RBD) IgG antibodies and cellular immunogenicity was assessed as CD4+/CD8 + T cell responses. Results: A total of 668 participants were vaccinated (332 aged 18–60 years and 336 aged > 60 years) including 75 who received homologous booster doses. Vaccination was well tolerated with no vaccine-related SAEs. Solicited and unsolicited AEs were mainly mild to moderate and resolved spontaneously. Both age groups demonstrated robust immune responses as neutralizing antibodies or RBD-binding IgG, after two doses, with lower titers in the older age group than the younger adults. Neither group achieved levels observed in human convalescent sera (HCS), but did equal or surpass HCS levels following homologous booster doses. Following CVnCoV vaccination, robust SARS-CoV-2 S-protein-specific CD4 + T-cell responses were observed in both age groups with CD8 + T-cell responses in some individuals, consistent with observations in convalescing COVID-19 patients after natural infection. Conclusions: We confirmed that two 12 µg doses of CVnCoV had an acceptable safety profile, and induced robust immune responses. Marked humoral immune responses to homologous boosters suggest two doses had induced immune memory.

This first-in-human study (NCT03032588), conducted in Belgium, evaluated a new inactivated poliovirus vaccines (IPV) candidate based on Sabin poliovirus strains grown on the high-yield PER.C6® cell line. Healthy adults (N = 32) were randomized (1:1) to receive a single dose of PER.C6-based Sabin-IPV (sIPV, 15:35:112.5 DU/dose) or conventional Salk-IPV (cIPV, 40:8:32 DU/dose). Reactogenicity was assessed up to 7 days after vaccination, immunogenicity 28 days after vaccination, and safety up to 6 months after vaccination. Solicited adverse events (AEs) were mild to moderate, no changes of concern in vital signs or safety laboratory values were observed, and no severe AEs (SAEs) or vaccine-related unsolicited AEs were reported after vaccination. A trend to more frequent solicited AEs after sIPV than after cIPV administration was observed. Most participants had preexisting neutralizing antibodies against poliovirus types (titer ≥8), which were strongly boosted by sIPV. Post-vaccination geometric mean titers were high (≥12,000) and similar across the two vaccination groups. Only participants with very high preexisting antibody levels did not show a vaccine-induced response, defined in seropositive participants as a 4-fold titer increase. The 10 initially seronegative (titer

Abstract Differences in innate immune ‘imprinting’ between vaccine adjuvants may mediate dissimilar effects on the quantity/quality of persisting adaptive responses. We compared antibody avidity maturation, antibody/memory B cell/CD4+ T cell response durability, and recall responses to non-adjuvanted fractional-dose antigen administered 1-year post-immunization (Day [D]360), between hepatitis B vaccines containing Adjuvant System (AS)01B, AS01E, AS03, AS04, or Alum (NCT00805389). Both the antibody and B cell levels ranked similarly (AS01B/E/AS03 > AS04 > Alum) at peak response, at D360, and following their increases post-antigen recall (D390). Proportions of high-avidity antibodies increased post-dose 2 across all groups and persisted at D360, but avidity maturation appeared to be more strongly promoted by AS vs. Alum. Post-antigen recall, frequencies of subjects with high-avidity antibodies increased only markedly in the AS groups. Among the AS, total antibody responses were lowest for AS04. However, proportions of high-avidity antibodies were similar between groups, suggesting that MPL in AS04 contributes to avidity maturation. Specific combinations of immunoenhancers in the AS, regardless of their individual nature, increase antibody persistence and avidity maturation.

Background: V-306 is a virus-like particle-based vaccine candidate displaying respiratory syncytial virus (RSV) F site II protein mimetics (FsIIm) as an antigenic epitope. Methods: This was a randomized, placebo-controlled, double-blind, dose-escalating, first-in-human study, conducted in 60 women aged 18–45 years. Twenty subjects per cohort (15 vaccine and five placebo) received two V-306 intramuscular administrations on Days 0 and 56 at 15 µg, 50 µg, or 150 µg. Safety and immunogenicity were assessed after each vaccination and for 1 year in total. Results: V-306 was safe and well tolerated at all dose levels, with no increase in reactogenicity and unsolicited adverse events between the first and second administrations. At 50 µg and 150 µg, V-306 induced an increase in FsIIm-specific immunoglobulin G (IgG) titers, which lasted at least 4 months. This did not translate into an increase in RSV-neutralizing antibody titers, which were already high at baseline. No increase in the anti-F protein-specific IgG titers was observed, which were also high in most subjects at baseline due to past natural infections. Conclusions: V-306 was safe and well-tolerated. Future modifications of the vaccine and assay conditions will likely improve the results of vaccination.

OVX836 is a recombinant protein-based vaccine targeting the highly conserved influenza nucleoprotein (NP), which aims to confer a broad-spectrum protection against influenza. In a Phase 1 study, OVX836, administered intramuscularly, has been found safe and immunogenic. The 90µg and 180µg dose levels were selected to be further evaluated in this randomized, monocenter, reference-controlled (Influvac Tetra™: quadrivalent seasonal influenza subunit vaccine), parallel group, double-blind, Phase 2a study in 300 healthy volunteers, aged 18-65 years, during the 2019/2020 flu season. Safety, influenza-like illness episodes (ILI; based on the Flu-PRO® questionnaire) and immunogenicity were assessed up to 180 days post-vaccination. OVX836 was safe and presented a reactogenicity profile similar to Influvac Tetra. It induced a significant increase in terms of NP-specific interferon-gamma (IFNγ) spot forming cells (SFCs), NP-specific CD4+ T-cells (essentially polyfunctional cells) and anti-NP IgG responses. OVX836 was superior to Influvac Tetra for all immunological parameters related to NP, and the 180µg dose was significantly superior to the 90µg dose for SFCs and CD4+ T-cells expressing IFNγ. Both the CD4+ T-cell and the anti-NP IgG responses persisted up to Day 180. An efficacy signal was observed with OVX836 at 180µg through reduction of ILI episodes occurring during the flu season as of 14 days post-vaccination. In conclusion, these results encourage further clinical evaluation of OVX836 in order to confirm the signal of efficacy on ILIs and/or laboratory-confirmed influenza cases. NCT04192500 (https://clinicaltrials.gov/ct2/show/study/NCT04192500)

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

Hepatitis C virus (HCV) is highly variable and transmits through infected blood to establish a chronic liver infection in the majority of patients. Our knowledge on the infectivity of clinical HCV strains is hampered by the lack of in vitro cell culture systems that support efficient viral replication. We and others have reported that HCV can associate with and infect immune cells and may thereby evade host immune surveillance and elimination. To evaluate whether B cells play a role in HCV transmission, we assessed the ability of B cells and sera from recent (

A multicomponent vaccine has been developed to reduce the frequency of acute exacerbations of COPD associated with non-typeable Haemophilus influenzae (NTHi) and Moraxella catarrhalis (Mcat) infections, containing NTHi (PD and PE-PilA) and Mcat (UspA2) surface proteins. In a randomised, observer-blind, placebo-controlled study with two steps (NCT02547974), the investigational vaccine had good immunogenicity and no safety concerns were identified. In step 2, 90 adults aged 50–71 years with smoking history received two doses 60 days apart of one of two AS01E-adjuvanted formulations containing 10 µg of each antigen (10–10-AS01) or 10 µg NTHi antigens and 3.3 µg UspA2 (10–3-AS01), or placebo. Long-term persistence of antigen-specific humoral antibodies was assessed in 81 participants during 3 years of follow-up after the initial 14-month study (NCT03201211).Antigen-specific antibody concentrations were measured in blood samples taken every 6 months. Safety monitoring evaluated serious adverse events (SAEs) and potential immune-mediated disease (pIMD).Immune responses against NTHi antigens persisted up to 4 years post-vaccination. For PD, PE and PilA, at each follow-up time point, adjusted antibody geometric mean concentrations (GMCs) were higher (non-overlapping 95% confidence intervals [CIs]) in the vaccine groups versus placebo and versus pre-vaccination. Antibody GMC point estimates were higher with 10–3-AS01 than with 10–10-AS01. For UspA2, 95% CIs included 1 for GMC ratios of 10–10-AS01 or 10–3-AS01 to placebo at each time point. During follow-up, SAEs were reported in nine (11.1%) participants, one of which was fatal (lung cancer, 607 days after second 10–10-AS01 dose). One non-serious pIMD, trigeminal neuralgia, was reported 771 days after second 10–3-AS01 dose. The SAEs and pIMD were considered not related to vaccination.Immune responses against NTHi antigens persisted for 4 years after two-dose vaccination with the investigational NTHi-Mcat vaccine. There was no persistent response against the Mcat antigen. No safety concerns were identified during the long-term follow-up.

Here, we report a randomized multicenter phase III trial assessing the lot-to-lot consistency of the 2014–2015 Northern Hemisphere quadrivalent split-virion inactivated influenza vaccine (IIV4; Sanofi Pasteur) and comparing its immunogenicity and safety with that of trivalent inactivated influenza vaccine (IIV3) in younger and older adults (EudraCT no. 2014-000785-21). Younger (18–60 y, n = 1114) and older (>60 y, n = 1111) adults were randomized 2:2:2:1:1 to receive a single dose of one of three lots of IIV4, the licensed IIV3 containing the B Yamagata lineage strain, or an investigational IIV3 containing the B Victoria lineage strain. Post-vaccination (day 21) hemagglutination inhibition antibody titers were equivalent for the three IIV4 lots. For the pooled IIV4s vs. IIV3, hemagglutination inhibition antibody titers were also non-inferior for the A strains, non-inferior for the B strain when present in the comparator IIV3, and superior for the B strain lineage when absent from the comparator IIV3. For all vaccine strains, seroprotection rates were ≥98% in younger adults and ≥90% in older adults. IIV4 also increased seroneutralizing antibody titers against all three vaccine strains of influenza. All vaccines were well tolerated, with no safety concerns identified. Solicited injection-site reactions were similar for IIV4 and IIV3 and mostly grade 1 and transient. This study showed that in younger and older adults, IIV4 had a similar safety profile as the licensed IIV3 and that including a second B strain lineage in IIV4 provided superior immunogenicity for the added B strain without affecting the immunogenicity of the three IIV3 strains.

Despite high vaccination coverage worldwide, pertussis has re-emerged in many countries. This randomized, controlled, observer-blind phase I study and extension study in Belgium (March 2012–June 2015) assessed safety and immunogenicity of investigational acellular pertussis vaccines containing genetically detoxified pertussis toxin (PT) (NCT01529645; NCT02382913). 420 healthy adults (average age: 26.8 ± 5.5 years, 60% female) were randomized to 1 of 10 vaccine groups: 3 investigational aP vaccines (containing pertussis antigens PT, filamentous hemagglutinin [FHA] and pertactin [PRN] at different dosages), 6 investigational TdaP (additionally containing tetanus toxoid [TT] and diphtheria toxoid [DT]), and 1 TdaP comparator containing chemically inactivated PT. Antibody responses were evaluated on days 1, 8, 30, 180, 365, and approximately 3 years post-booster vaccination. Cell-mediated immune responses and PT neutralization were evaluated in a subset of participants in pre-selected groups. Local and systemic adverse events (AEs), and unsolicited AEs were collected through day 7 and 30, respectively; serious AEs and AEs leading to study withdrawal were collected through day 365 post-vaccination. Antibody responses against pertussis antigens peaked at day 30 post-vaccination and then declined but remained above baseline level at approximately 3 years post-vaccination. Responses to FHA and PRN were correlated to antigen dose. Antibody responses specific to PT, toxin neutralization activity and persistence induced by investigational formulations were similar or significantly higher than the licensed vaccine, despite lower PT doses. Of 15 serious AEs, none were considered vaccination-related; 1 led to study withdrawal (premature labor, day 364; aP4 group). This study confirmed the potential benefits of genetically detoxified PT antigen. All investigational study formulations were well tolerated.

Vaccination is the most effective and well-tolerated method of conferring long-term protection against hepatitis A and B viruses (HAV; HBV). Long-term studies are required to characterize the duration of protection and need for boosters. Following primary immunization of 150 and 157 healthy adults with 3-doses of combined hepatitis A/hepatitis B vaccine (HAB; Twinrix™, GSK Vaccines, Belgium) at 0-1-6 months in 2 separate studies, we measured vaccine-induced antibody persistence against HAV and HBV annually for 20 y (Study A: NCT01000324; Study B: NCT01037114). Subjects with circulating anti-HAV antibodies < 15 mIU/mL or with anti-hepatitis B surface antigen < 10 mIU/mL were offered an additional monovalent hepatitis A and/or B vaccine dose (Havrix™/Engerix™-B, GSK Vaccines, Belgium). Applying the immunogenicity results from these studies, mathematical modeling predicted long-term persistence. After 20 y, 18 and 25 subjects in studies A and B, respectively, comprised the long-term according-to-protocol cohort for immunogenicity; 100% and 96.0% retained anti-HAV antibodies ≥ 15 mIU/mL, respectively; 94.4% and 92.0% had anti-HBs antibodies ≥ 10 mIU/mL, respectively. Between Years 16–20, 4 subjects who received a challenge dose of monovalent hepatitis A vaccine (N = 2) or hepatitis B vaccine (N = 2), all mounted a strong anamnestic response suggestive of immune memory despite low antibody levels. Mathematical modeling predicts that 40 y after vaccination ≥ 97% vaccinees will maintain anti-HAV ≥ 15 mIU/mL and ≥ 50% vaccinees will retain anti-HBs ≥ 10 mIU/mL. Immunogenicity data confirm that primary immunization with 3-doses of HAB induces persisting anti-HAV and anti-HBs specific antibodies in most adults for up to 20 y; mathematical modeling predicts even longer-term protection.

Systems biology has the potential to identify gene signatures associated with vaccine immunogenicity and protective efficacy. The main objective of this study was to identify optimal postvaccination time points for evaluating peripheral blood RNA expression profiles in relation to vaccine immunogenicity and potential efficacy in recipients of the candidate tuberculosis vaccine M72/AS01. In this phase II open-label study (NCT01669096; https://clinicaltrials.gov/), healthy Bacillus Calmette–Guérin-primed, HIV-negative adults were administered two doses (30 days apart) of M72/AS01. Twenty subjects completed the study and 18 subjects received two doses. Blood samples were collected pre-dose 1, pre-dose 2, and 1, 7, 10, 14, 17, and 30 days post-dose 2. RNA expression in whole blood (WB) and peripheral blood mononuclear cells (PBMCs) was quantified using microarray technology. Serum interferon-gamma responses and M72-specific CD4+ T cell responses to vaccination, and the observed safety profile were similar to previous trials. Two different approaches were utilized to analyze the RNA expression data. First, a kinetic analysis of RNA expression changes using blood transcription modules revealed early (1 day post-dose 2) activation of several pathways related to innate immune activation, both in WB and PBMC. Second, using a previously identified gene signature as a classifier, optimal postvaccination time points were identified. Since M72/AS01 efficacy remains to be established, a PBMC-derived gene signature associated with the protective efficacy of a similarly adjuvanted candidate malaria vaccine was used as a proxy for this purpose. This approach was based on the assumption that the AS01 adjuvant used in both studies could induce shared innate immune pathways. Subjects were classified as gene signature positive (GS+) or gene signature negative (GS−). Assignments of subjects to GS+ or GS− groups were confirmed by significant differences in RNA expression of the gene signature genes in PBMCs at 14 days post-dose 2 relative to prevaccination and in WB samples at 7, 10, 14, and 17 days post-dose 2 relative to prevaccination. Hence, in comparison with a prevaccination, 7, 10, 14, and 17 days postvaccination appeared to be suitable time points for identifying potentially clinically relevant transcriptome responses to M72/AS01 in WB samples.

To elucidate the role of innate responses in vaccine immunogenicity, we compared early responses to hepatitis B virus (HBV) surface antigen (HBsAg) combined with different Adjuvant Systems (AS) in healthy HBV-naïve adults, and included these parameters in multi-parametric models of adaptive responses. A total of 291 participants aged 18–45 years were randomized 1:1:1:1:1 to receive HBsAg with AS01B, AS01E, AS03, AS04, or Alum/Al(OH)3 at days 0 and 30 (ClinicalTrials.gov: NCT00805389). Blood protein, cellular, and mRNA innate responses were assessed at early time-points and up to 7 days after vaccination, and used with reactogenicity symptoms in linear regression analyses evaluating their correlation with HBs-specific CD4+ T-cell and antibody responses at day 44. All AS induced transient innate responses, including interleukin (IL)-6 and C-reactive protein (CRP), mostly peaking at 24 h post-vaccination and subsiding to baseline within 1–3 days. After the second but not the first injection, median interferon (IFN)-γ levels were increased in the AS01B group, and IFN-γ-inducible protein-10 levels and IFN-inducible genes upregulated in the AS01 and AS03 groups. No distinct marker or signature was specific to one particular AS. Innate profiles were comparable between AS01B, AS01E, and AS03 groups, and between AS04 and Alum groups. AS group rankings within adaptive and innate response levels and reactogenicity prevalence were similar (AS01B ≥ AS01E > AS03 > AS04 > Alum), suggesting an association between magnitudes of inflammatory and vaccine responses. Modeling revealed associations between adaptive responses and specific traits of the innate response post-dose 2 (activation of the IFN-signaling pathway, CRP and IL-6 responses). In conclusion, the ability of AS01 and AS03 to enhance adaptive responses to co-administered HBsAg is likely linked to their capacity to activate innate immunity, particularly the IFN-signaling pathway.

In the context of early vaccine trials aimed at evaluating the safety profile of novel vaccines, abnormal haematological values, such as neutropenia, are often reported. It is therefore important to evaluate how these trials should be planned not to miss potentially important safety signals, but also to understand the implications and the clinical relevance.We report and discuss the results from five clinical trials (two with a new Shigella vaccine in the early stage of clinical development and three with licensed vaccines) where the absolute neutrophil counts (ANC) were evaluated before and after vaccination. Additionally, we have performed a systematic review of the literature on cases of neutropenia reported during vaccine trials to discuss our results in a more general context.Both in our clinical trials and in the literature review, several cases of neutropenia have been reported, in the first two weeks after vaccination. However, neutropenia was generally transient and had a benign clinical outcome, after vaccination with either multiple novel candidates or well-known licensed vaccines. Additionally, the vaccine recipients with neutropenia frequently had lower baseline ANC than non-neutropenic vaccinees. In many instances neutropenia occurred in subjects of African descent, known to have lower ANC compared to western populations.It is important to include ANC and other haematological tests in early vaccine trials to identify potential safety signals. Post-vaccination neutropenia is not uncommon, generally transient and clinically benign, but many vaccine trials do not have a sampling schedule that allows its detection. Given ethnic variability in the level of circulating neutrophils, normal ranges taking into account ethnicity should be used for determination of trial inclusion/exclusion criteria and classification of neutropenia related adverse events.ClinicalTrials.gov NCT02017899, NCT02034500, NCT01771367, NCT01765413, NCT02523287.

Avian influenza continues to circulate and remains a global health threat not least because of the associated high mortality. In this study antibody persistence, booster vaccine response and cross-clade immune response between two influenza A(H5N1) vaccines were compared. Participants aged over 18-years who had previously been immunized with a clade 1, A/Vietnam vaccine were re-immunized at 6-months with 7.5 μg of the homologous strain or at 22-months with a clade 2, alum-adjuvanted, A/Indonesia vaccine. Blood sampled at 6, 15 and 22-months after the primary course was used to assess antibody persistence. Antibody concentrations 6-months after primary immunisation with either A/Vietnam vaccine 30 μg alum-adjuvanted vaccine or 7.5 μg dose vaccine were lower than 21-days after the primary course and waned further with time. Re-immunization with the clade 2, 30 μg alum-adjuvanted vaccine confirmed cross-clade reactogenicity. Antibody cross-reactivity between A(H5N1) clades suggests that in principle a prime-boost vaccination strategy may provide both early protection at the start of a pandemic and improved antibody responses to specific vaccination once available.ClinicalTrials.gov NCT00415129.

In the 1970s and 1980s it became increasingly clear that blood transfusions could induce a form of chronic hepatitis that could not be ascribed to any of the viruses known to cause liver inflammation. In 1989, the hepatitis C virus (HCV) was discovered and found to be the major causative agent of these infections. Because of its narrow ropism, the in vivo study of this virus was, especially in the early days, limited to the chimpanzee. In the past decade, several alternative animal models have been created. In this review we review these novel animal models and their contribution to our current understanding of the biology of HCV.

A highly efficacious pre-erythrocytic stage vaccine would be an important tool for the control and elimination of malaria but is currently unavailable. High-level protection in humans can be achieved by experimental immunization with Plasmodium falciparum sporozoites attenuated by radiation or under anti-malarial drug coverage. Immunization with genetically attenuated parasites (GAP) would be an attractive alternative approach. In this study, we present data on safety and protective efficacy using sporozoites with deletions of two genes, that is the newly identified b9 and slarp, which govern independent and critical processes for successful liver-stage development. In the rodent malaria model, PbΔb9ΔslarpGAP was completely attenuated showing no breakthrough infections while efficiently inducing high-level protection. The human PfΔb9ΔslarpGAP generated without drug resistance markers were infective to human hepatocytes in vitro and to humanized mice engrafted with human hepatocytes in vivo but completely aborted development after infection. These findings support the clinical development of a PfΔb9ΔslarpSPZ vaccine.

The nature of protective immune responses elicited by immunization with the candidate malaria vaccine RTS,S is still incompletely understood. Antibody levels correlate with protection against malaria infection, but considerable variation in outcome is unexplained (e.g., children may experience malaria despite high anti-circumsporozoite [CS] titers).We measured the avidity index (AI) of the anti-CS antibodies raised in subgroup of 5-17 month old children in Kenya who were vaccinated with three doses of RTS,S/AS01E between March and August 2007. We evaluated the association between the AI and the subsequent risk of clinical malaria. We selected 19 cases (i.e., with clinical malaria) and 42 controls (i.e., without clinical malaria), matching for anti-CS antibody levels and malaria exposure. We assessed their sera collected 1 month after the third dose of the vaccine, in March 2008 (range 4-10 months after the third vaccine), and at 12 months after the third vaccine dose. The mean AI was 45.2 (95% CI: 42.4 to 48.1), 45.3 (95% CI: 41.4 to 49.1) and 46.2 (95% CI; 43.2 to 49.3) at 1 month, in March 2008 (4-10 months), and at 12 months after the third vaccination, respectively (p = 0.9 by ANOVA test for variation over time). The AI was not associated with protection from clinical malaria (OR = 0.90; 95% CI: 0.49 to 1.66; p = 0.74). The AI was higher in children with high malaria exposure, as measured using the weighted local prevalence of malaria, compared to those with low malaria exposure at 1 month post dose 3 (p = 0.035).Our data suggest that in RTS,S/AS01E-vaccinated children residing in malaria endemic countries, the avidity of anti-circumsporozoite antibodies, as measured using an elution ELISA method, was not associated with protection from clinical malaria. Prior natural malaria exposure might have primed the response to RTS,S/AS01E vaccination.

Mucosal antibodies harboring various antiviral activities may best protect mucosal surfaces against early HIV-1 entry at mucosal sites and they should be ideally induced by prophylactic HIV-1 vaccines for optimal prevention of sexually transmitted HIV-1. A phase I, double-blind, randomized, placebo-controlled trial was conducted in twenty-four healthy HIV-uninfected young women. The study objectives were to assess the safety, tolerability and immunogenicity of virosomes harboring surface HIV-1 gp41-derived P1 lipidated peptides (MYM-V101). Participants received placebo or MYM-V101 vaccine at 10 μg/dose or 50 μg/dose intramuscularly at week 0 and 8, and intranasally at week 16 and 24. MYM-V101 was safe and well-tolerated at both doses administered by the intramuscular and intranasal routes, with the majority of subjects remaining free of local and general symptoms. P1-specific serum IgGs and IgAs were induced in all high dose recipients after the first injection. After the last vaccination, vaginal and rectal P1-specific IgGs could be detected in all high dose recipients. Approximately 63% and 43% of the low and high dose recipients were respectively tested positive for vaginal P1-IgAs, while 29% of the subjects from the high dose group tested positive for rectal IgAs. Serum samples had total specific IgG and IgA antibody concentrations ≥ 0.4 μg/mL, while mucosal samples were usually below 0.01 μg/mL. Vaginal secretions from MYM-V101 vaccinated subjects were inhibiting HIV-1 transcytosis but had no detectable neutralizing activity. P1-specific Th1 responses could not be detected on PBMC. This study demonstrates the excellent safety and tolerability of MYM-V101, eliciting systemic and mucosal antibodies in the majority of subjects. Vaccine-induced mucosal anti-gp41 antibodies toward conserved gp41 motifs were harboring HIV-1 transcytosis inhibition activity and may contribute to reduce sexually-transmitted HIV-1.ClinicalTrials.gov NCT01084343.

A distinctive feature of HCV is that its life cycle depends on lipoprotein metabolism. Viral morphogenesis and secretion follow the very low-density lipoprotein (VLDL) biogenesis pathway and, consequently, infectious HCV in the serum is associated with triglyceride-rich lipoproteins (TRL). Lipoprotein lipase (LPL) hydrolyzes TRL within chylomicrons and VLDL but, independently of its catalytic activity, it has a bridging activity, mediating the hepatic uptake of chylomicrons and VLDL remnants. We previously showed that exogenously added LPL increases HCV binding to hepatoma cells by acting as a bridge between virus-associated lipoproteins and cell surface heparan sulfate, while simultaneously decreasing infection levels. We show here that LPL efficiently inhibits cell infection with two HCV strains produced in hepatoma cells or in primary human hepatocytes transplanted into uPA-SCID mice with fully functional human ApoB-lipoprotein profiles. Viruses produced in vitro or in vivo were separated on iodixanol gradients into low and higher density populations, and the infection of Huh 7.5 cells by both virus populations was inhibited by LPL. The effect of LPL depended on its enzymatic activity. However, the lipase inhibitor tetrahydrolipstatin restored only a minor part of HCV infectivity, suggesting an important role of the LPL bridging function in the inhibition of infection. We followed HCV cell entry by immunoelectron microscopy with anti-envelope and anti-core antibodies. These analyses demonstrated the internalization of virus particles into hepatoma cells and their presence in intracellular vesicles and associated with lipid droplets. In the presence of LPL, HCV was retained at the cell surface. We conclude that LPL efficiently inhibits HCV infection by acting on TRL associated with HCV particles through mechanisms involving its lipolytic function, but mostly its bridging function. These mechanisms lead to immobilization of the virus at the cell surface. HCV-associated lipoproteins may therefore be a promising target for the development of new therapeutic approaches.

With the advent of subgenomic hepatitis C virus (HCV) replicons, studies of the intracellular steps of the viral replication cycle became possible. These RNAs are capable of self-amplification in cultured human hepatoma cells, but save for the genotype 2a isolate JFH-1, efficient replication of these HCV RNAs requires replication enhancing mutations (REMs), previously also called cell culture adaptive mutations. These mutations cluster primarily in the central region of non-structural protein 5A (NS5A), but may also reside in the NS3 helicase domain or at a distinct position in NS4B. Most efficient replication has been achieved by combining REMs residing in NS3 with distinct REMs located in NS4B or NS5A. However, in spite of efficient replication of HCV genomes containing such mutations, they do not support production of infectious virus particles. By using the genotype 1b isolate Con1, in this study we show that REMs interfere with HCV assembly. Strongest impairment of virus formation was found with REMs located in the NS3 helicase (E1202G and T1280I) as well as NS5A (S2204R), whereas a highly adaptive REM in NS4B still allowed virus production although relative levels of core release were also reduced. We also show that cells transfected with the Con1 wild type genome or the genome containing the REM in NS4B release HCV particles that are infectious both in cell culture and in vivo. Our data provide an explanation for the in vitro and in vivo attenuation of cell culture adapted HCV genomes and may open new avenues for the development of fully competent culture systems covering the therapeutically most relevant HCV genotypes.

BackgroundThe availability of H5N1 vaccines that can elicit a broad cross-protective immunity against different currently circulating clade 2 H5N1 viruses is a pre-requisite for the development of a successful pre-pandemic vaccination strategy. In this regard, it has recently been shown that adjuvantation of a recombinant clade 1 H5N1 inactivated split-virion vaccine with an oil-in-water emulsion-based adjuvant system also promoted cross-immunity against a recent clade 2 H5N1 isolate (A/Indonesia/5/2005, subclade 2.1). Here we further analyse the cross-protective potential of the vaccine against two other recent clade 2 isolates (A/turkey/Turkey/1/2005 and A/Anhui/1/2005 which are, as defined by WHO, representatives of subclades 2.2 and 2.3 respectively).Methods and findingsTwo doses of the recombinant A/Vietnam/1194/2004 (H5N1, clade 1) vaccine were administered 21 days apart to volunteers aged 18-60 years. We studied the cross-clade immunogenicity of the lowest antigen dose (3.8 microg haemagglutinin) given with (N = 20) or without adjuvant (N = 20). Immune responses were assessed at 21 days following the first and second vaccine doses and at 6 months following first vaccination. Vaccination with two doses of 3.8 microg of the adjuvanted vaccine induced four-fold neutralising seroconversion rates in 85% of subjects against A/turkey/Turkey/1/2005 (subclade 2.2) and 75% of subjects against A/Anhui/1/2005 (subclade 2.3) recombinant strains. There was no response induced against these strains in the non-adjuvanted group. At 6 months following vaccination, 70% and 60% of subjects retained neutralising antibodies against the recombinant subclade 2.2 and 2.3 strains, respectively and 40% of subjects retained antibodies against the recombinant subclade 2.1 A/Indonesia/5/2005 strain.ConclusionsIn addition to antigen dose-sparing, adjuvantation of inactivated split H5N1 vaccine promotes broad and persistent cross-clade immunity which is a pre-requisite for a pre-pandemic vaccine.Trial registrationClinicalTrials.gov NCT00309634.