I studied biochemistry and obtained a PhD at the University of Lyon, France, in 2000, working on DNA immunization as a therapeutic approach against chronic Hepatitis B Virus infection, in collaboration with the Institut National de la Sante et Recherche Medicale (INSERM), Lyon, France. I proceeded to work on novel vaccine development and cellular immunity against Hepatitis C Virus chronic infection at the Biomedical Primate Research Center, The Netherlands.
In 2007 I joined the Jenner Institute at the University of Oxford as a senior immunologist, to work on improvements of vaccine vectors against malaria. I then moved to the Oxford Vaccine Group in 2010, bringing my expertise of viral vectored vaccine platform and preclinical research, and became Associate Professor in Vaccinology. I started, developed and led the Novel Vaccine Development team involved in the creation, design, preclinical and early clinical studies of new and improved vaccines against bacterial diseases and infectious diseases affecting children. My research activities, funded by the Medical research Council, Innovate UK, the Oxford Biomedical Research Centre, and several charities, include the conception, design, pre-clinical and early clinical development of new and improved vaccines against bacterial infections such as caspular group B meningococcus, plague, Q fever, enteric fever, pertussis, gonorrhoea and Respiratory Syncitial Virus.
In 2021 I joined the University of Surrey as Professor of Vaccinology.
Areas of specialism
In the media
My research interest are in vaccine development from design and preclinical studies to early clinical development. I have expertise in different vaccine platforms and approaches and seek to adapt those to the target pathogen and to the target population, with a focus on OneHealth, anti-microbial resistance and vulnerable populations (children, elderly and low-and-middle-income countries).
PhD project led by Dr Elma Tchilian, The Pirbright Institute
PhD project led by Dr Chris Netherton, The Pirbright Institute
Vaccine development against gonorrhoea, led by Prof Calman MacLennan, The Jenner Institute, University of Oxford
Indicators of esteem
Jenner Institute Investigator (since 2014)
Visiting scientist at The University of Oxford (since 2021)
We evaluated the adjuvant effect of a modified glycoform of lipopolysaccharide (LPS) (LgtB-LpxL1) compared to that of the nonmodified glycoform Lpxl1 serogroup B meningococcal H44/76 native outer membrane vesicles (nOMVs) on immune responses to vaccination with the recombinant meningococcal protein, rPorA, tetanus toxoid, or meningococcal serogroup C capsular polysaccharide. We used LgtB-LpxL1 LPS because the disruption of the lgtB gene, which results in the exposure of N -acetylglucosamine-galactose-glucose residues in the LPS outer core, has been shown to enhance the activation of human dendritic cells in vitro . The responses were compared to those of a monophosphoryl lipid A (MPL)-based adjuvant and to an aluminum hydroxide suspension. The nOMVs induced blood serum IgG responses against each of the three antigens comparable to those obtained with MPL or aluminum salt. However, nOMVs elicited (i) a lower IgG1/IgG2a ratio against rPorA and (ii) serum bactericidal antibody titers superior to those achieved with aluminum salt, reaching similar titers to those obtained with MPL. Similarly, bactericidal antibody titers induced by immunization with meningococcal serogroup C polysaccharide and nOMVs were similar to those obtained using MPL but were better than those with aluminum salt. Immunization with tetanus toxoid and nOMVs resulted in tetanus toxoid-specific IgG responses similar to those obtained when adjuvanted with aluminum salt. These results highlight the potential utility of meningococcal LpxL1 LPS-containing nOMVs as an adjuvant for recombinant meningococcal protein vaccines and suggest their possible use with a variety of other antigens.
Treatment of severe COVID-19 is currently limited by clinical heterogeneity and incomplete description of specific immune biomarkers. We present here a comprehensive multi-omic blood atlas for patients with varying COVID-19 severity in an integrated comparison with influenza and sepsis patients versus healthy volunteers. We identify immune signatures and correlates of host response. Hallmarks of disease severity involved cells, their inflammatory mediators and networks, including progenitor cells and specific myeloid and lymphocyte subsets, features of the immune repertoire, acute phase response, metabolism and coagulation. Persisting immune activation involving AP-1/p38MAPK was a specific feature of COVID-19. The plasma proteome enabled sub-phenotyping into patient clusters, predictive of severity and outcome. Systems-based integrative analyses including tensor and matrix decomposition of all modalities revealed feature groupings linked with severity and specificity compared to influenza and sepsis. Our approach and blood atlas will support future drug development, clinical trial design and personalized medicine approaches for COVID-19. [Display omitted] •Blood atlas delineating innate and adaptive immune dysregulation in COVID-19•Shared and specific immune signatures of COVID-19, influenza and all cause sepsis•Multi-omic immune profiling differentiates hospitalised patient severity in COVID-19•Immune activation and proliferation involving AP-1/p38MAPK associated with COVID-19 A multi-omic analysis of patient blood samples reveals both similarities and specific features of COVID-19 when compared with samples obtained from sepsis or influenza patients which could yield better targetted therapies for severe COVID-19.
Neisseria meningitidis outer membrane vesicle (OMV) vaccines are safe and provide strain-specific protection against invasive meningococcal disease (IMD) primarily by inducing serum bactericidal antibodies against the outer membrane proteins (OMP). To design broader coverage vaccines, knowledge of the immunogenicity of all the antigens contained in OMVs is needed. In a Phase I clinical trial, an investigational meningococcal OMV vaccine, MenPF1, made from a meningococcus genetically modified to constitutively express the iron-regulated FetA induced bactericidal responses to both the PorA and the FetA antigen present in the OMP. Using peripheral blood mononuclear cells collected from this trial, we analyzed the kinetics of and relationships between IgG, IgA, and IgM B cell responses against recombinant PorA and FetA, including (i) antibody-secreting cells, (ii) memory B cells, and (iii) functional antibody responses (opsonophagocytic and bactericidal activities). Following MenPF1vaccination, PorA-specific IgG secreting cell responses were detected in up to 77% of participants and FetA-specific responses in up to 36%. Memory B cell responses to the vaccine were low or absent and mainly detected in participants who had evidence of preexisting immunity ( = 0.0069). Similarly, FetA-specific antibody titers and bactericidal activity increased in participants with preexisting immunity and is consistent with the idea that immune responses are elicited to minor antigens during asymptomatic carriage, which can be boosted by OMV vaccines. Neisseria meningitidis outer membrane vesicles (OMV) are a component of the capsular group B meningococcal vaccine 4CMenB (Bexsero) and have been shown to induce 30% efficacy against gonococcal infection. They are composed of multiple antigens and are considered an interesting delivery platform for vaccines against several bacterial diseases. However, the protective antibody response after two or three doses of OMV-based meningococcal vaccines appears short-lived. We explored the B cell response induced to a dominant and a subdominant antigen in a meningococcal OMV vaccine in a clinical trial and showed that immune responses are elicited to minor antigens. However, memory B cell responses to the OMV were low or absent and mainly detected in participants who had evidence of preexisting immunity against the antigens. Failure to induce a strong B cell response may be linked with the low persistence of protective responses.
The pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) might be curtailed by vaccination. We assessed the safety, reactogenicity, and immunogenicity of a viral vectored coronavirus vaccine that expresses the spike protein of SARS-CoV-2. We did a phase 1/2, single-blind, randomised controlled trial in five trial sites in the UK of a chimpanzee adenovirus-vectored vaccine (ChAdOx1 nCoV-19) expressing the SARS-CoV-2 spike protein compared with a meningococcal conjugate vaccine (MenACWY) as control. Healthy adults aged 18-55 years with no history of laboratory confirmed SARS-CoV-2 infection or of COVID-19-like symptoms were randomly assigned (1:1) to receive ChAdOx1 nCoV-19 at a dose of 5 × 10 viral particles or MenACWY as a single intramuscular injection. A protocol amendment in two of the five sites allowed prophylactic paracetamol to be administered before vaccination. Ten participants assigned to a non-randomised, unblinded ChAdOx1 nCoV-19 prime-boost group received a two-dose schedule, with the booster vaccine administered 28 days after the first dose. Humoral responses at baseline and following vaccination were assessed using a standardised total IgG ELISA against trimeric SARS-CoV-2 spike protein, a muliplexed immunoassay, three live SARS-CoV-2 neutralisation assays (a 50% plaque reduction neutralisation assay [PRNT ]; a microneutralisation assay [MNA , MNA , and MNA ]; and Marburg VN), and a pseudovirus neutralisation assay. Cellular responses were assessed using an ex-vivo interferon-γ enzyme-linked immunospot assay. The co-primary outcomes are to assess efficacy, as measured by cases of symptomatic virologically confirmed COVID-19, and safety, as measured by the occurrence of serious adverse events. Analyses were done by group allocation in participants who received the vaccine. Safety was assessed over 28 days after vaccination. Here, we report the preliminary findings on safety, reactogenicity, and cellular and humoral immune responses. The study is ongoing, and was registered at ISRCTN, 15281137, and ClinicalTrials.gov, NCT04324606. Between April 23 and May 21, 2020, 1077 participants were enrolled and assigned to receive either ChAdOx1 nCoV-19 (n=543) or MenACWY (n=534), ten of whom were enrolled in the non-randomised ChAdOx1 nCoV-19 prime-boost group. Local and systemic reactions were more common in the ChAdOx1 nCoV-19 group and many were reduced by use of prophylactic paracetamol, including pain, feeling feverish, chills, muscle ache, headache, and malaise (all p
COVID-19 vaccine supply shortages are causing concerns about compromised immunity in some countries as the interval between the first and second dose becomes longer. Conversely, countries with no supply constraints are considering administering a third dose. We assessed the persistence of immunogenicity after a single dose of ChAdOx1 nCoV-19 (AZD1222), immunity after an extended interval (44–45 weeks) between the first and second dose, and response to a third dose as a booster given 28–38 weeks after the second dose. In this substudy, volunteers aged 18–55 years who were enrolled in the phase 1/2 (COV001) controlled trial in the UK and had received either a single dose or two doses of 5 × 1010 viral particles were invited back for vaccination. Here we report the reactogenicity and immunogenicity of a delayed second dose (44–45 weeks after first dose) or a third dose of the vaccine (28–38 weeks after second dose). Data from volunteers aged 18–55 years who were enrolled in either the phase 1/2 (COV001) or phase 2/3 (COV002), single-blinded, randomised controlled trials of ChAdOx1 nCoV-19 and who had previously received a single dose or two doses of 5 × 1010 viral particles are used for comparison purposes. COV001 is registered with ClinicalTrials.gov, NCT04324606, and ISRCTN, 15281137, and COV002 is registered with ClinicalTrials.gov, NCT04400838, and ISRCTN, 15281137, and both are continuing but not recruiting. Between March 11 and 21, 2021, 90 participants were enrolled in the third-dose boost substudy, of whom 80 (89%) were assessable for reactogenicity, 75 (83%) were assessable for evaluation of antibodies, and 15 (17%) were assessable for T-cells responses. The two-dose cohort comprised 321 participants who had reactogenicity data (with prime-boost interval of 8–12 weeks: 267 [83%] of 321; 15–25 weeks: 24 [7%]; or 44–45 weeks: 30 [9%]) and 261 who had immunogenicity data (interval of 8–12 weeks: 115 [44%] of 261; 15–25 weeks: 116 [44%]; and 44–45 weeks: 30 [11%]). 480 participants from the single-dose cohort were assessable for immunogenicity up to 44–45 weeks after vaccination. Antibody titres after a single dose measured approximately 320 days after vaccination remained higher than the titres measured at baseline (geometric mean titre of 66·00 ELISA units [EUs; 95% CI 47·83–91·08] vs 1·75 EUs [1·60–1·93]). 32 participants received a late second dose of vaccine 44–45 weeks after the first dose, of whom 30 were included in immunogenicity and reactogenicity analyses. Antibody titres were higher 28 days after vaccination in those with a longer interval between first and second dose than for those with a short interval (median total IgG titre: 923 EUs [IQR 525–1764] with an 8–12 week interval; 1860 EUs [917–4934] with a 15–25 week interval; and 3738 EUs [1824–6625] with a 44–45 week interval). Among participants who received a third dose of vaccine, antibody titres (measured in 73 [81%] participants for whom samples were available) were significantly higher 28 days after a third dose (median total IgG titre: 3746 EUs [IQR 2047–6420]) than 28 days after a second dose (median 1792 EUs [IQR 899–4634]; Wilcoxon signed rank test p=0·0043). T-cell responses were also boosted after a third dose (median response increased from 200 spot forming units [SFUs] per million peripheral blood mononuclear cells [PBMCs; IQR 127–389] immediately before the third dose to 399 SFUs per milion PBMCs [314–662] by day 28 after the third dose; Wilcoxon signed rank test p=0·012). Reactogenicity after a late second dose or a third dose was lower than reactogenicity after a first dose. An extended interval before the second dose of ChAdOx1 nCoV-19 leads to increased antibody titres. A third dose of ChAdOx1 nCoV-19 induces antibodies to a level that correlates with high efficacy after second dose and boosts T-cell responses. UK Research and Innovation, Engineering and Physical Sciences Research Council, National Institute for Health Research, Coalition for Epidemic Preparedness Innovations, National Institute for Health Research Oxford Biomedical Research Centre, Chinese Academy of Medical Sciences Innovation Fund for Medical Science, Thames Valley and South Midlands NIHR Clinical Research Network, AstraZeneca, and Wellcome.
Older adults (aged ≥70 years) are at increased risk of severe disease and death if they develop COVID-19 and are therefore a priority for immunisation should an efficacious vaccine be developed. Immunogenicity of vaccines is often worse in older adults as a result of immunosenescence. We have reported the immunogenicity of a novel chimpanzee adenovirus-vectored vaccine, ChAdOx1 nCoV-19 (AZD1222), in young adults, and now describe the safety and immunogenicity of this vaccine in a wider range of participants, including adults aged 70 years and older. In this report of the phase 2 component of a single-blind, randomised, controlled, phase 2/3 trial (COV002), healthy adults aged 18 years and older were enrolled at two UK clinical research facilities, in an age-escalation manner, into 18-55 years, 56-69 years, and 70 years and older immunogenicity subgroups. Participants were eligible if they did not have severe or uncontrolled medical comorbidities or a high frailty score (if aged ≥65 years). First, participants were recruited to a low-dose cohort, and within each age group, participants were randomly assigned to receive either intramuscular ChAdOx1 nCoV-19 (2·2 × 10 virus particles) or a control vaccine, MenACWY, using block randomisation and stratified by age and dose group and study site, using the following ratios: in the 18-55 years group, 1:1 to either two doses of ChAdOx1 nCoV-19 or two doses of MenACWY; in the 56-69 years group, 3:1:3:1 to one dose of ChAdOx1 nCoV-19, one dose of MenACWY, two doses of ChAdOx1 nCoV-19, or two doses of MenACWY; and in the 70 years and older, 5:1:5:1 to one dose of ChAdOx1 nCoV-19, one dose of MenACWY, two doses of ChAdOx1 nCoV-19, or two doses of MenACWY. Prime-booster regimens were given 28 days apart. Participants were then recruited to the standard-dose cohort (3·5-6·5 × 10 virus particles of ChAdOx1 nCoV-19) and the same randomisation procedures were followed, except the 18-55 years group was assigned in a 5:1 ratio to two doses of ChAdOx1 nCoV-19 or two doses of MenACWY. Participants and investigators, but not staff administering the vaccine, were masked to vaccine allocation. The specific objectives of this report were to assess the safety and humoral and cellular immunogenicity of a single-dose and two-dose schedule in adults older than 55 years. Humoral responses at baseline and after each vaccination until 1 year after the booster were assessed using an in-house standardised ELISA, a multiplex immunoassay, and a live severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) microneutralisation assay (MNA ). Cellular responses were assessed using an ex-vivo IFN-γ enzyme-linked immunospot assay. The coprimary outcomes of the trial were efficacy, as measured by the number of cases of symptomatic, virologically confirmed COVID-19, and safety, as measured by the occurrence of serious adverse events. Analyses were by group allocation in participants who received the vaccine. Here, we report the preliminary findings on safety, reactogenicity, and cellular and humoral immune responses. This study is ongoing and is registered with ClinicalTrials.gov, NCT04400838, and ISRCTN, 15281137. Between May 30 and Aug 8, 2020, 560 participants were enrolled: 160 aged 18-55 years (100 assigned to ChAdOx1 nCoV-19, 60 assigned to MenACWY), 160 aged 56-69 years (120 assigned to ChAdOx1 nCoV-19: 40 assigned to MenACWY), and 240 aged 70 years and older (200 assigned to ChAdOx1 nCoV-19: 40 assigned to MenACWY). Seven participants did not receive the boost dose of their assigned two-dose regimen, one participant received the incorrect vaccine, and three were excluded from immunogenicity analyses due to incorrectly labelled samples. 280 (50%) of 552 analysable participants were female. Local and systemic reactions were more common in participants given ChAdOx1 nCoV-19 than in those given the control vaccine, and similar in nature to those previously reported (injection-site pain, feeling feverish, muscle ache, headache), but were less common in older adults (aged ≥56 years) than younger adults. In those receiving two standard doses of ChAdOx1 nCoV-19, after the prime vaccination local reactions were reported in 43 (88%) of 49 participants in the 18-55 years group, 22 (73%) of 30 in the 56-69 years group, and 30 (61%) of 49 in the 70 years and older group, and systemic reactions in 42 (86%) participants in the 18-55 years group, 23 (77%) in the 56-69 years group, and 32 (65%) in the 70 years and older group. As of Oct 26, 2020, 13 serious adverse events occurred during the study period, none of which were considered to be related to either study vaccine. In participants who received two doses of vaccine, median anti-spike SARS-CoV-2 IgG responses 28 days after the boost dose were similar across the three age cohorts (standard-dose groups: 18-55 years, 20 713 arbitrary units [AU]/mL [IQR 13 898-33 550], n=39; 56-69 years, 16 170 AU/mL [10 233-40 353], n=26; and ≥70 years 17 561 AU/mL [9705-37 796], n=47; p=0·68). Neutralising antibody titres after a boost dose were similar across all age groups (median MNA at day 42 in the standard-dose groups: 18-55 years, 193 [IQR 113-238], n=39; 56-69 years, 144 [119-347], n=20; and ≥70 years, 161 [73-323], n=47; p=0·40). By 14 days after the boost dose, 208 (>99%) of 209 boosted participants had neutralising antibody responses. T-cell responses peaked at day 14 after a single standard dose of ChAdOx1 nCoV-19 (18-55 years: median 1187 spot-forming cells [SFCs] per million peripheral blood mononuclear cells [IQR 841-2428], n=24; 56-69 years: 797 SFCs [383-1817], n=29; and ≥70 years: 977 SFCs [458-1914], n=48). ChAdOx1 nCoV-19 appears to be better tolerated in older adults than in younger adults and has similar immunogenicity across all age groups after a boost dose. Further assessment of the efficacy of this vaccine is warranted in all age groups and individuals with comorbidities. UK Research and Innovation, National Institutes for Health Research (NIHR), Coalition for Epidemic Preparedness Innovations, NIHR Oxford Biomedical Research Centre, Thames Valley and South Midlands NIHR Clinical Research Network, and AstraZeneca.
The ChAdOx1 nCoV-19 (AZD1222) vaccine has been approved for emergency use by the UK regulatory authority, Medicines and Healthcare products Regulatory Agency, with a regimen of two standard doses given with an interval of 4-12 weeks. The planned roll-out in the UK will involve vaccinating people in high-risk categories with their first dose immediately, and delivering the second dose 12 weeks later. Here, we provide both a further prespecified pooled analysis of trials of ChAdOx1 nCoV-19 and exploratory analyses of the impact on immunogenicity and efficacy of extending the interval between priming and booster doses. In addition, we show the immunogenicity and protection afforded by the first dose, before a booster dose has been offered. We present data from three single-blind randomised controlled trials-one phase 1/2 study in the UK (COV001), one phase 2/3 study in the UK (COV002), and a phase 3 study in Brazil (COV003)-and one double-blind phase 1/2 study in South Africa (COV005). As previously described, individuals 18 years and older were randomly assigned 1:1 to receive two standard doses of ChAdOx1 nCoV-19 (5 × 10 viral particles) or a control vaccine or saline placebo. In the UK trial, a subset of participants received a lower dose (2·2 × 10 viral particles) of the ChAdOx1 nCoV-19 for the first dose. The primary outcome was virologically confirmed symptomatic COVID-19 disease, defined as a nucleic acid amplification test (NAAT)-positive swab combined with at least one qualifying symptom (fever ≥37·8°C, cough, shortness of breath, or anosmia or ageusia) more than 14 days after the second dose. Secondary efficacy analyses included cases occuring at least 22 days after the first dose. Antibody responses measured by immunoassay and by pseudovirus neutralisation were exploratory outcomes. All cases of COVID-19 with a NAAT-positive swab were adjudicated for inclusion in the analysis by a masked independent endpoint review committee. The primary analysis included all participants who were SARS-CoV-2 N protein seronegative at baseline, had had at least 14 days of follow-up after the second dose, and had no evidence of previous SARS-CoV-2 infection from NAAT swabs. Safety was assessed in all participants who received at least one dose. The four trials are registered at ISRCTN89951424 (COV003) and ClinicalTrials.gov, NCT04324606 (COV001), NCT04400838 (COV002), and NCT04444674 (COV005). Between April 23 and Dec 6, 2020, 24 422 participants were recruited and vaccinated across the four studies, of whom 17 178 were included in the primary analysis (8597 receiving ChAdOx1 nCoV-19 and 8581 receiving control vaccine). The data cutoff for these analyses was Dec 7, 2020. 332 NAAT-positive infections met the primary endpoint of symptomatic infection more than 14 days after the second dose. Overall vaccine efficacy more than 14 days after the second dose was 66·7% (95% CI 57·4-74·0), with 84 (1·0%) cases in the 8597 participants in the ChAdOx1 nCoV-19 group and 248 (2·9%) in the 8581 participants in the control group. There were no hospital admissions for COVID-19 in the ChAdOx1 nCoV-19 group after the initial 21-day exclusion period, and 15 in the control group. 108 (0·9%) of 12 282 participants in the ChAdOx1 nCoV-19 group and 127 (1·1%) of 11 962 participants in the control group had serious adverse events. There were seven deaths considered unrelated to vaccination (two in the ChAdOx1 nCov-19 group and five in the control group), including one COVID-19-related death in one participant in the control group. Exploratory analyses showed that vaccine efficacy after a single standard dose of vaccine from day 22 to day 90 after vaccination was 76·0% (59·3-85·9). Our modelling analysis indicated that protection did not wane during this initial 3-month period. Similarly, antibody levels were maintained during this period with minimal waning by day 90 (geometric mean ratio [GMR] 0·66 [95% CI 0·59-0·74]). In the participants who received two standard doses, after the second dose, efficacy was higher in those with a longer prime-boost interval (vaccine efficacy 81·3% [95% CI 60·3-91·2] at ≥12 weeks) than in those with a short interval (vaccine efficacy 55·1% [33·0-69·9] at
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of Coronavirus Disease 2019 (COVID-19), has caused a global pandemic, and safe, effective vaccines are urgently needed . Strong, Th1-skewed T cell responses can drive protective humoral and cell-mediated immune responses and might reduce the potential for disease enhancement . Cytotoxic T cells clear virus-infected host cells and contribute to control of infection . Studies of patients infected with SARS-CoV-2 have suggested a protective role for both humoral and cell-mediated immune responses in recovery from COVID-19 (refs. ). ChAdOx1 nCoV-19 (AZD1222) is a candidate SARS-CoV-2 vaccine comprising a replication-deficient simian adenovirus expressing full-length SARS-CoV-2 spike protein. We recently reported preliminary safety and immunogenicity data from a phase 1/2 trial of the ChAdOx1 nCoV-19 vaccine (NCT04400838) given as either a one- or two-dose regimen. The vaccine was tolerated, with induction of neutralizing antibodies and antigen-specific T cells against the SARS-CoV-2 spike protein. Here we describe, in detail, exploratory analyses of the immune responses in adults, aged 18-55 years, up to 8 weeks after vaccination with a single dose of ChAdOx1 nCoV-19 in this trial, demonstrating an induction of a Th1-biased response characterized by interferon-γ and tumor necrosis factor-α cytokine secretion by CD4 T cells and antibody production predominantly of IgG1 and IgG3 subclasses. CD8 T cells, of monofunctional, polyfunctional and cytotoxic phenotypes, were also induced. Taken together, these results suggest a favorable immune profile induced by ChAdOx1 nCoV-19 vaccine, supporting the progression of this vaccine candidate to ongoing phase 2/3 trials to assess vaccine efficacy.
Abstract Background Viral vectors, including adenovirus (Ad) and modified vaccinia Ankara (MVA), have gained increasing attention as vaccine platforms in recent years due to their capacity to express antigens from a wide array of pathogens, their rapid induction of humoral and cellular protective immune responses, and their relatively low production costs. In particular, the chimpanzee Ad vector, ChAdOx1, has taken centre stage as a leading COVID-19 vaccine candidate. However, despite mounting data, both clinical and pre-clinical, demonstrating effective induction of adaptive immune responses, the innate immune signals that precede the protective responses that make these vectors attractive vaccine platforms remain poorly understood. Results In this study, a mouse immunisation model was used to evaluate whole blood gene expression changes 24 h after either a single dose or heterologous prime-boost regimen of an Ad and/or MVA vaccine. We demonstrate through comparative analysis of Ad vectors encoding different antigens that a transgene product-specific gene signature can be discerned from the vector-induced transcriptional response. Expression of genes involved in TLR2 stimulation and γδ T cell and natural killer cell activation were induced after a single dose of Ad, while MVA led to greater expression of type I interferon genes. The order of prime-boost combinations was found to influence the magnitude of the gene expression changes, with MVA/Ad eliciting greater transcriptional perturbation than Ad/MVA. Contrasting the two regimens revealed significant enrichment of epigenetic regulation pathways and augmented expression of MHC class I and II molecules associated with MVA/Ad. Conclusion These data demonstrate that the order in which vaccines from heterologous prime-boost regimens are administered leads to distinct transcriptional responses and may shape the immune response induced by such combinations. The characterisation of early vaccine-induce responses strengthens our understanding of viral vector vaccine mechanisms of action ahead of their characterisation in human clinical trials and are a valuable resource to inform the pre-clinical design of appropriate vaccine constructs for emerging infectious diseases.
Despite the existence of a highly efficient yellow fever vaccine, yellow fever reemergence throughout Africa and the Americas has put 900 million people in 47 countries at risk of contracting the disease. Although the vaccine has been key to controlling yellow fever epidemics, its live-attenuated nature comes with a range of contraindications that prompts advising against its administration to pregnant and lactating women, immunocompromised individuals, and those with hypersensitivity to chicken egg proteins. Additionally, large outbreaks have highlighted problems with insufficient vaccine supply, whereby manufacturers rely on slow traditional manufacturing processes that prevent them from ramping up production. These limitations have contributed to an inadequate control of yellow fever and have favored the pursuit of novel yellow fever vaccine candidates that aim to circumvent the licensed vaccine's restrictions. Here, we review the live-attenuated vaccine's limitations and explore the epitome of a yellow fever vaccine, whilst scrutinizing next-generation vaccine candidates.
More than 190 vaccines are currently in development to prevent infection by the novel severe acute respiratory syndrome coronavirus 2. Animal studies suggest that while neutralizing antibodies against the viral spike protein may correlate with protection, additional antibody functions may also be important in preventing infection. Previously, we reported early immunogenicity and safety outcomes of a viral vector coronavirus vaccine, ChAdOx1 nCoV-19 (AZD1222), in a single-blinded phase 1/2 randomized controlled trial of healthy adults aged 18-55 years ( NCT04324606 ). Now we describe safety and exploratory humoral and cellular immunogenicity of the vaccine, from subgroups of volunteers in that trial, who were subsequently allocated to receive a homologous full-dose (SD/SD D56; n = 20) or half-dose (SD/LD D56; n = 32) ChAdOx1 booster vaccine 56 d following prime vaccination. Previously reported immunogenicity data from the open-label 28-d interval prime-boost group (SD/SD D28; n = 10) are also presented to facilitate comparison. Additionally, we describe volunteers boosted with the comparator vaccine (MenACWY; n = 10). In this interim report, we demonstrate that a booster dose of ChAdOx1 nCoV-19 is safe and better tolerated than priming doses. Using a systems serology approach we also demonstrate that anti-spike neutralizing antibody titers, as well as Fc-mediated functional antibody responses, including antibody-dependent neutrophil/monocyte phagocytosis, complement activation and natural killer cell activation, are substantially enhanced by a booster dose of vaccine. A booster dose of vaccine induced stronger antibody responses than a dose-sparing half-dose boost, although the magnitude of T cell responses did not increase with either boost dose. These data support the two-dose vaccine regime that is now being evaluated in phase 3 clinical trials.
Neisseria meningitidis is a major cause of meningitis and septicaemia. A MenB vaccine (4CMenB) was licensed by the European Medicines Agency in January 2013. Here we describe the blood transcriptome and proteome following infant immunisations with or without concomitant 4CMenB, to gain insight into the molecular mechanisms underlying post‐vaccination reactogenicity and immunogenicity. Infants were randomised to receive control immunisations (PCV13 and DTaP‐IPV‐Hib) with or without 4CMenB at 2 and 4 months of age. Blood gene expression and plasma proteins were measured prior to, then 4 h, 24 h, 3 days or 7 days post‐vaccination. 4CMenB vaccination was associated with increased expression of ENTPD7 and increased concentrations of 4 plasma proteins: CRP, G‐CSF, IL‐1RA and IL‐6. Post‐vaccination fever was associated with increased expression of SELL , involved in neutrophil recruitment. A murine model dissecting the vaccine components found the concomitant regimen to be associated with increased gene perturbation compared with 4CMenB vaccine alone with enhancement of pathways such as interleukin‐3, ‐5 and GM‐CSF signalling. Finally, we present transcriptomic profiles predictive of immunological and febrile responses following 4CMenB vaccine. A randomised clinical trial evaluates transcriptomic and proteomic profiles following infant concomitant 4CMenB vaccination, compared with control vaccines alone. A novel framework is provided for both understanding and predicting vaccine immunogenicity and reactogenicity.
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global pandemic in 2020. Testing is crucial for mitigating public health and economic effects. Serology is considered key to population-level surveillance and potentially individual-level risk assessment. However, immunoassay performance has not been compared on large, identical sample sets. We aimed to investigate the performance of four high-throughput commercial SARS-CoV-2 antibody immunoassays and a novel 384-well ELISA. We did a head-to-head assessment of SARS-CoV-2 IgG assay (Abbott, Chicago, IL, USA), LIAISON SARS-CoV-2 S1/S2 IgG assay (DiaSorin, Saluggia, Italy), Elecsys Anti-SARS-CoV-2 assay (Roche, Basel, Switzerland), SARS-CoV-2 Total assay (Siemens, Munich, Germany), and a novel 384-well ELISA (the Oxford immunoassay). We derived sensitivity and specificity from 976 pre-pandemic blood samples (collected between Sept 4, 2014, and Oct 4, 2016) and 536 blood samples from patients with laboratory-confirmed SARS-CoV-2 infection, collected at least 20 days post symptom onset (collected between Feb 1, 2020, and May 31, 2020). Receiver operating characteristic (ROC) curves were used to assess assay thresholds. At the manufacturers' thresholds, for the Abbott assay sensitivity was 92·7% (95% CI 90·2–94·8) and specificity was 99·9% (99·4–100%); for the DiaSorin assay sensitivity was 96·2% (94·2–97·7) and specificity was 98·9% (98·0–99·4); for the Oxford immunoassay sensitivity was 99·1% (97·8–99·7) and specificity was 99·0% (98·1–99·5); for the Roche assay sensitivity was 97·2% (95·4–98·4) and specificity was 99·8% (99·3–100); and for the Siemens assay sensitivity was 98·1% (96·6–99·1) and specificity was 99·9% (99·4–100%). All assays achieved a sensitivity of at least 98% with thresholds optimised to achieve a specificity of at least 98% on samples taken 30 days or more post symptom onset. Four commercial, widely available assays and a scalable 384-well ELISA can be used for SARS-CoV-2 serological testing to achieve sensitivity and specificity of at least 98%. The Siemens assay and Oxford immunoassay achieved these metrics without further optimisation. This benchmark study in immunoassay assessment should enable refinements of testing strategies and the best use of serological testing resource to benefit individuals and population health. Public Health England and UK National Institute for Health Research.
Adenoviral vectors are being developed as vaccines against infectious agents and tumour-associated antigens, because of their ability to induce cellular immunity. However, the protection afforded in animal models has not easily translated into primates and clinical trials, underlying the need for improving adenoviral vaccines-induced immunogenicity. A Toll-like receptor signalling molecule, TRAM, was assessed for its ability to modify the immune responses induced by an adenovirus-based vaccine. Different adenovirus vectors either expressing TRAM alone or co-expressing TRAM along with a model antigen were constructed. The modification of T-cell and antibody responses induced by TRAM was assessed in vivo in mice and in primates. Co-expression of TRAM and an antigen from adenoviruses increased the transgene-specific CD8+ T cell responses in mice. Similar effects were seen when a TRAM expressing virus was co-administered with the antigen-expressing adenovirus. However, in primate studies, co-administration of a TRAM expressing adenovirus with a vaccine expressing the ME-TRAP malaria antigen had no significant effect on the immune responses. While these results support the idea that modification of innate immune signalling by genetic vectors modifies immunogenicity, they also emphasise the difficulty in generalising results from rodents into primates, where the regulatory pathway may be different to that in mice.
The development of an effective vaccine against respiratory syncytial virus (RSV) has been hampered by major difficulties that occurred in the 1960s when a formalin-inactivated vaccine led to increased severity of RSV disease after acquisition of the virus in the RSV season after vaccination. Recent renewed efforts to develop a vaccine have resulted in about 38 candidate vaccines and monoclonal antibodies now in clinical development. The target populations for effective vaccination are varied and include neonates, young children, pregnant women, and older adults. The reasons for susceptibility to infection in each of these groups may be different and, therefore, could require different vaccine types for induction of protective immune responses, adding a further challenge for vaccine development. Here, we review the current knowledge of RSV vaccine development for these target populations and propose a view and rationale for prioritizing RSV vaccine development.
The capsular group B meningococcal (MenB) four component vaccine (4CMenB) has been licensed for the prevention of invasive disease caused by MenB. The vaccine causes fever in infants, particularly when given in combination (concomitant) with other routinely-administered vaccines (routine), such as the standard diphtheria, tetanus, pertussis (DTP)-containing vaccine. To assess the suitability of a mouse immunisation model to study this phenomenon, we monitored temperature in mice after a second dose of routine vaccines, with or without 4CMenB, and compared the results with those in humans. Using this mouse model, we explored the reactogenicity of 4CMenB components by measuring changes in temperature, cytokines, and gene expression induced by 4CMenB, one of its components, wild-type or attenuated endotoxin outer membrane vesicles (OMVs), or lipopolysaccharide (LPS). A significant rise (p
•Long-term S. aureus experimental gut colonisation can be established in two mouse strains.•Colonisation results in detectable serum IgG1 anti-S. aureus antibodies.•Responses to individual antigens induced by colonisation vary within genetically identical mice.•Immune responses against EapH2 are generated in some, but not other, colonised mice.•Natural priming modified the response to subsequent vaccination against EapH2. S. aureus is a pathogen to which individuals are exposed shortly after birth, with immune responses to S. aureus increasing during childhood. There is marked heterogeneity between the anti- S. aureus immune responses of different humans, the basis of which is not fully understood. To investigate development of anti-S. aureus immune responses, we studied S. aureus colonised mice under controlled conditions. Mice were either acquired colonised from breeding colonies, or experimentally colonised by exposure to a cage environment which had been sprayed with a S. aureus suspension. Colonisation was monitored by sequential stool sampling, and immunoglobulin levels against both whole fixed S. aureus and individual S. aureus antigens quantified. The immunological impact of colonisation on subsequent vaccination was investigated. Colonised BALB/c and BL/6 mice develop serum anti- S. aureus cell surface IgG1 antibodies. Responses were proportional to the cumulative S. aureus bioburden in the mice, and were higher in BALB/c mice, which have higher colonisation levels, than in C57BL/6 animals. We observed marked variation in the induction of anti-cell surface antibodies, even in genetically identical mice experimentally colonised with the same S. aureus clone. Heterogeneity was also evident when monitoring immune responses to the secreted S. aureus protein EapH2. Approximately 50% of colonised mice developed anti-EapH2 responses (responders); in other mice, responses were not significantly different to those in uncolonised mice (non-responders). Following vaccination with a replication deficient adenovirus expressing EapH2, less anti-EapH2 antibody was generated in non-responder than responder animals. In genetically identical mice, S. aureus colonisation results in all-or-nothing antibody responses against some antigens, including EapH2. For antigens involved in colonisation success by microbes, apparently stochastic early immune responses may impact both vaccine responses and the establishment of an animal-specific microbiome.
•We investigated the impact of vaccination with the S. aureus proteins EapH1 and EapH2.•Intranasal vaccination with adenovirus expressing EapH1 and H2 reduced S. aureus carriage.•EapH1/H2 vaccination also reduced bacterial load following i.v. injection of S. aureus.•EapH1/H2 vaccination may offer an antibiotic-independent way of reducing S. aureus carriage. There is a need for an efficacious vaccine reducing infections due to Staphylococcus aureus, a common cause of community and hospital infection. Infecting organisms originate from S. aureus populations colonising the nares and bowel. Antimicrobials are widely used to transiently reduce S. aureus colonisation prior to surgery, a practice which is selecting for resistant S. aureus isolates. S. aureus secretes multiple proteins, including the protease inhibitors extracellular adhesion protein homologue 1 and 2 (EapH1 and EapH2). Mice were vaccinated intramuscularly or intranasally with Adenovirus serotype 5 and Modified Vaccinia Ankara viral vectors expressing EapH1 and EapH2 proteins, or with control viruses. Using murine S. aureus colonisation models, we monitored S. aureus colonisation by sequential stool sampling. Monitoring of S. aureus invasive disease after intravenous challenge was performed using bacterial load and abscess numbers in the kidney. Intramuscular vaccination with Adenovirus serotype 5 and Modified Vaccinia Ankara viral vectors expressing EapH1 and EapH2 proteins significantly reduces bacterial recovery in the murine renal abscess model of infection, but the magnitude of the effect is small. A single intranasal vaccination with an adenoviral vaccine expressing these proteins reduced S. aureus gastrointestinal (GI) tract colonisation. Vaccination against EapH1 / EapH2 proteins may offer an antibiotic independent way to reduce S. aureus colonisation, as well as contributing to protection against S. aureus invasive disease.
Introduction: The emergence of the Zika virus (ZIKV) in Latin America in 2015-2016 led to an expeditious search for vaccine candidates, with a DNA-based candidate having progressed to Phase II. However, several features of ZIKV infection and epidemiology are not understood, which may be key to maximizing efficacy and ensuring safety of ZIKV vaccines. Areas covered: Conceivable problems related to vaccine development and policy include: (1) paucity of diagnostics to satisfactorily discriminate between past ZIKV and dengue virus (DENV) exposure; (2) insufficient knowledge of the mechanisms of ZIKV neurovirulence, amongst other unknowns in the biology of this infection, is particularly relevant from a vaccine safety perspective; and (3) the potential for disease enhancement, as observed with DENV infection and vaccine. Expert opinion: Vaccine candidates that entered phase I/II trials have demonstrated protection in naïve animal models, while ZIKV epidemics occurred in populations that had encountered DENV before. The resulting cross-reactive antibodies pose problems for reliable serologic diagnostic assays, and for the potential of disease enhancement. The alleged neurological complications also warrant further exploration in order to reassure regulators of the safety profile of these vaccines in target populations. These research aspects should be an integral part of the efforts to develop a vaccine.
•Serogroup X meningococci (MenX) has caused localized outbreaks in Africa in the last decade, and vaccines are in development.•To inform vaccine development we investigated the serum antibody responses against MenX in individuals living in an area affected by a MenX outbreak during 2007 in Togo, West Africa.•We found that dependent on age, 29–34% of Togolese study participants showed serum bactericidal antibody against the MenX strain; significantly higher than controls from the U.K. and Burkina Faso. Capsular group X N. meningitidis (MenX) has emerged as a cause of localized disease outbreaks in sub-Saharan Africa, but the human immune response following exposure to MenX antigens is poorly described. We therefore assessed the natural immunity against MenX in individuals who were living in an area affected by a MenX outbreak during 2007 in Togo, West Africa. During 2009, 300 healthy individuals (100 aged 3–5 years, 100 aged 13–19 years and 100 aged 20–25 years) were included in the study, and serum responses were compared with sera from age-matched controls from the U.K. and Burkina Faso. MenX serum bactericidal antibody (SBA) was measured using rabbit complement, and antibodies against MenX polysaccharide (XPS) and outer membrane vesicles (XOMVs) were quantified by ELISA. The proportion of Togolese individuals with an SBA titer of ≥8 against the MenX strain was 29% (95% confidence interval (CI) 18–41) among those aged 3–5 years, 34% (95% CI 9–60) among those aged 13–19 years and 32% (95% CI 24–40) among those aged 20–25 years. These were significantly higher than observed in the control populations from the U.K (range 13–16%) and Burkina Faso (range 2–6%). In Togolese individuals, the concentration of serum IgG against XPS was higher among the two older age groups as compared to the youngest age group. Antibody concentrations against MenX PS correlated significantly with SBA titers. This supports further development of a MenX PS based conjugate vaccine. Further studies are needed to verify the ability of MenX PS to induce SBA in humans.
One fifth to one quarter of the human population is asymptomatically, naturally and persistently colonised by Staphylococcus aureus. Observational human studies indicate that although the whole population is intermittently exposed, some individuals lose S. aureus rapidly. Others become persistent carriers, as assessed by nasal cultures, with many individuals colonised for decades. Current animal models of S. aureus colonisation are expensive and normally require antibiotics. Importantly, these animal models have not yet contributed to our poor understanding of the dichotomy in human colonisation status. Here, we identify a single strain of S. aureus found to be persistently colonising the gastrointestinal tract of BALB/c mice. Phylogenetic analyses suggest it diverged from a human ST15 lineage in the recent past. We show that murine carriage of this organism occurs in the bowel and nares, is acquired early in life, and can persist for months. Importantly, we observe the development of persistent and non-persistent gastrointestinal carriage states in genetically identical mice. We developed a needle- and antibiotic-free model in which we readily induced S. aureus colonisation of the gastrointestinal tract experimentally by environmental exposure. Using our experimental model, impact of adaptive immunity on S. aureus colonisation could be assessed. Vaccine efficacy to eliminate colonisation could also be investigated using this model.
Powder-injectors use gas propulsion to deposit lyophilised drug or vaccine particles in the epidermal and sub epidermal layers of the skin. We prepared dry-powder (Tg = 45.2 ± 0.5°C) microparticles (58.1 μm) of a MenY-CRM197 glyconjugate vaccine (0.5% wt.) for intradermal needle-free powder injection (NFPI). SFD used ultrasound atomisation of the liquid vaccine-containing excipient feed, followed by lyophilisation above the glass transition temperature (Tg' = - 29.9 ± 0.3°C). This resulted in robust particles (density~ 0.53 ±0.09 g/cm3) with a narrow volume size distribution (mean diameter 58.1 μm, and span = 1.2), and an impact parameter (ρvr ~ 11.5 kg/m·s) sufficient to breach the Stratum corneum (sc). The trehalose, manitol, dextran (10 kDa), dextran (150 kDa) formulation, or TMDD (3:3:3:1), protected the MenY-CRM197 glyconjugate during SFD with minimal loss, no detectable chemical degradation or physical aggregation. In a capsular group Y Neisseria meningitidis serum bactericidal assay (SBA) with human serum complement, the needle free vaccine, which contained no alum adjuvant, induced functional protective antibody responses in vivo of similar magnitude to the conventional vaccine injected by hypodermic needle and syringe and containing alum adjuvant. These results demonstrate that needle-free vaccination is both technically and immunologically valid, and could be considered for vaccines in development.
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A persistent goal of vaccine development is the enhancement of the immunogenicity of antigens while maintaining safety. One strategy involves alteration of the presentation of the antigen by combining antigens with a multimeric scaffold. Multi-antigen vaccines are under development, and there are presently far more candidate antigens than antigen scaffolding strategies. This is potentially problematic, since prior immunity to a scaffold may inhibit immune responses to the antigen-scaffold combination. In this study, a series of domains from S. aureus which have been shown to crystallise into multimeric structures have been examined for their scaffolding potential. Of these domains, SAR1376, a 62 amino acid member of the 4-oxalocrotonate tautomerase (4-OT) family, was pro-immunogenic in mice when fused to a range of pathogen antigens from both S. aureus and P. falciparum, and delivered by either DNA vaccination, viral vector vaccines or as protein-in-adjuvant formulations. The adjuvant effect did not depend on enzymatic activity, but was abrogated by mutations disrupting the hexameric structure of the protein. We therefore propose that SAR1376, and perhaps other members of the 4-OT protein family, represent very small domains which can be fused to a wide range of antigens, enhancing immune responses against them.
MenB-4C is a meningococcal vaccine for the prevention of serogroup B disease. The vaccine contains factor H binding protein (FHbp) and three other antigens that can elicit serum bactericidal antibodies (SBA). For vaccine licensure, efficacy was inferred from the SBA responses against three antigen-specific indicator strains. The relation between those results and broad protection against circulating genetically diverse strains is not known. Twenty adults were immunized with two doses of MenB-4C given 1 to 2 months apart. SBA activity against 3 reference strains and 15 serogroup B test strains (6 from college outbreaks) was measured. Compared to the preimmunization titers, 70%, 95%, and 95% of subjects had ≥4-fold increases in the titers of anti-PorA P1.4, anti-NadA, and anti-FHbp antibodies against the reference strains, respectively. In contrast, only 25 to 45% of the subjects had ≥4-fold increases in responses to 10 of the 15 test strains, including 8 that expressed one to three of the antigens in the vaccine. At 1 month, the majority of subjects with
•Viral-vectored vaccines are expected to induce T-cell responses to sub-dominant epitopes.•Hierarchy of T-cell response is influenced by the timing of analysis after a single immunization.•Repeated homologous immunization reduces the breadth of T-cell response.•Heterologous prime-boost induces a modest increase of the subdominant responses. Viral-vectored vaccines are in clinical development for several infectious diseases where T-cell responses can mediate protection, and responses to sub-dominant epitopes is needed. Little is known about the influence of MVA or adenoviral vectors on the hierarchy of the dominant and sub-dominant T-cell epitopes. We investigated this aspect in mice using a malaria immunogen. Our results demonstrate that the T-cell hierarchy is influenced by the timing of analysis, rather than by the vector after a single immunization, with hierarchy changing over time. Repeated homologous immunization reduced the breadth of responses, while heterologous prime-boost induced the strongest response to the dominant epitope, albeit with only modest response to the sub-dominant epitopes.
Immune responses against multiple epitopes are required for the prevention of hepatitis C virus (HCV) infection, and the progression to phase I trials of candidates may be guided by comparative immunogenicity studies in non-human primates. Four vectors, DNA, SFV, human serotype 5 adenovirus (HuAd5) and Modified Vaccinia Ankara (MVA) poxvirus, all expressing hepatitis C virus Core, E1, E2 and NS3, were combined in three prime-boost regimen, and their ability to elicit immune responses against HCV antigens in rhesus macaques was explored and compared. All combinations induced specific T-cell immune responses, including high IFN-γ production. The group immunized with the SFV+MVA regimen elicited higher E2-specific responses as compared with the two other modalities, while animals receiving HuAd5 injections elicited lower IL-4 responses as compared with those receiving MVA. The IFN-γ responses to NS3 were remarkably similar between groups. Only the adenovirus induced envelope-specific antibody responses, but these failed to show neutralizing activity. Therefore, the two novel regimens failed to induce superior responses as compared with already existing HCV vaccine candidates. Differences were found in response to envelope proteins, but the relevance of these remain uncertain given the surprisingly poor correlation with immunogenicity data in chimpanzees, underlining the difficulty to predict efficacy from immunology studies.
Viral vectors are the vaccine platform of choice for many pathogens that have thwarted efforts towards control using conventional vaccine approaches. Although the STEP trial encumbered development of recombinant human adenovirus vectors only a few years ago, replication-deficient simian adenoviruses have since emerged as a crucial component of clinically effective prime-boost regimens. The vectors discussed here elicit functionally relevant cellular and humoral immune responses, at extremes of age and in diverse populations. The recent Ebola virus outbreak highlighted the utility of viral vectored vaccines in facilitating a rapid response to public health emergencies. Meanwhile, technological advances in manufacturing to support scale-up of viral vectored vaccines have helped to consolidate their position as a leading approach to tackling 'old' and emerging infections.
To develop and validate a sensitive and specific method of abscess enumeration and quantification in a preclinical model of Staphylococcus aureus infection. S. aureus infected murine kidneys were fixed in paraformaldehyde, impregnated with gadolinium, and embedded in agar blocks, which were subjected to 3D magnetic resonance microscopy on a 9.4T MRI scanner. Image analysis techniques were developed, which could identify and quantify abscesses. The result of this imaging was compared with histological examination. The impact of a S. aureus Sortase A vaccination regime was assessed using the technique. Up to 32 murine kidneys could be imaged in a single MRI run, yielding images with voxels of about 25 μm3. S. aureus abscesses could be readily identified in blinded analyses of the kidneys after 3 days of infection, with low inter-observer variability. Comparison with histological sections shows a striking correlation between the two techniques: all presumptive abscesses identified by MRI were confirmed histologically, and histology identified no abscesses not evident on MRI. In view of this, simulations were performed assuming that both MRI reconstruction, and histology examining all sections of the tissue, were fully sensitive and specific at abscess detection. This simulation showed that MRI provided more sensitive and precise estimates of abscess numbers and volume than histology, unless at least 5 histological sections are taken through the long axis of the kidney. We used the MRI technique described to investigate the impact of a S. aureus Sortase A vaccine. Post mortem MRI scanning of large batches of fixed organs has application in the preclinical assessment of S. aureus vaccines.
Staphylococcus aureus is a major cause of life-threatening bacterial infection. A significant risk factor for infection is nasal carriage. Previously, we reported spontaneous mutations during carriage associated with infection, including loss-of-function of the gene repressor of surface proteins ( rsp ). Here we use genomic screens, experimental assays, and molecular examination of rsp mutants from patients to understand how rsp is involved in infection; we find it has far-reaching effects on gene regulation. Paradoxically, rsp mutants exhibited attenuated toxicity and reduced disease severity early in experimental infection, without sacrificing the ability to cause abscesses and bloodstream infection. This work reveals a complex relationship between correlates of disease in the laboratory and in patients, demonstrating that life-threatening disease can be associated with reduced severity early in infection. Staphylococcus aureus is a major bacterial pathogen, which causes severe blood and tissue infections that frequently emerge by autoinfection with asymptomatically carried nose and skin populations. However, recent studies report that bloodstream isolates differ systematically from those found in the nose and skin, exhibiting reduced toxicity toward leukocytes. In two patients, an attenuated toxicity bloodstream infection evolved from an asymptomatically carried high-toxicity nasal strain by loss-of-function mutations in the gene encoding the transcription factor repressor of surface proteins ( rsp ). Here, we report that rsp knockout mutants lead to global transcriptional and proteomic reprofiling, and they exhibit the greatest signal in a genome-wide screen for genes influencing S. aureus survival in human cells. This effect is likely to be mediated in part via SSR 42, a long-noncoding RNA. We show that rsp controls SSR 42 expression, is induced by hydrogen peroxide, and is required for normal cytotoxicity and hemolytic activity. Rsp inactivation in laboratory- and bacteremia-derived mutants attenuates toxin production, but up-regulates other immune subversion proteins and reduces lethality during experimental infection. Crucially, inactivation of rsp preserves bacterial dissemination, because it affects neither formation of deep abscesses in mice nor survival in human blood. Thus, we have identified a spontaneously evolving, attenuated-cytotoxicity, nonhemolytic S. aureus phenotype, controlled by a pleiotropic transcriptional regulator/noncoding RNA virulence regulatory system, capable of causing S. aureus bloodstream infections. Such a phenotype could promote deep infection with limited early clinical manifestations, raising concerns that bacterial evolution within the human body may contribute to severe infection.
•There is marked variation in anti-S. aureus antibodies between healthy humans.•Serum IgG1 against S. aureus surface antigens declines as adults age.•High anti-S. aureus IgG3 levels bypass bacterial subversion of phagocyte function.•Properties of IgG subclasses should be considered in S. aureus vaccine development. Staphylococcus aureus is a pathogen which causes life-threatening infection, the incidence of which rises during adult life. This, together with the emergence of drug-resistant strains and the expansion of more susceptible elderly populations, represents the rationale for the ongoing development of S. aureus vaccines targeting adult populations. Humoral responses to S. aureus naturally develop early in life, influence susceptibility to infection, and potentially influence the effect of vaccination. Despite this, the nature of pre-existing anti-S. aureus antibodies in healthy adult populations is not fully characterised. Immunoglobulin levels against S. aureus surface antigens were measured by a filter membrane enzyme-linked immunosorbent assay using fixed ΔSpA S. aureus as an antigen in serum samples obtained from three clinical cohorts comprising 133 healthy adult volunteers from 19 to 65 years of age. Functional capacity of antibody was also assessed, using antibody-mediated attachment of FITC-stained S. aureus to differentiated HL-60 cells. Wide variation in the concentrations of immunoglobulins recognising S. aureus surface antigens was observed among individuals in all three cohorts. There was a decline of anti-S. aureus IgG1 with age, and a similar trend was observed in IgM, but not in IgA or other IgG sub-classes. Antibody mediated bacterial attachment to cells was associated with IgG1 and IgG3 concentrations in serum. The presence of SpA on the bacterial cell surface reduced antibody-mediated binding of bacteria to phagocytes in serum with low, but not high, levels of naturally occurring anti-S. aureus IgG3 antibodies. Naturally acquired immunoglobulin responses to S. aureus are heterogeneous in populations and their concentrations alter during adulthood. Elevated IgG1 or IgG3 titres against S. aureus enhance S. aureus recognition by phagocytosis and may be correlates of natural protection and/or vaccine efficacy in adult populations.
Neisseria meningitidis recruits host human complement inhibitors to its surface to down-regulate complement activation and enhance survival in blood. We have investigated whether such complement inhibitor binding occurs after vaccination with native outer membrane vesicles (nOMVs), and limits immunogenicity of such vaccines. To this end, nOMVs reactogenic lipopolysaccharide was detoxified by deletion of the lpxl1 gene (nOMVlpxl1). nOMVs unable to bind human complement factor H (hfH) were generated by additional deletions of the genes encoding factor H binding protein (fHbp) and neisserial surface protein A (NspA) (nOMVdis). Antibody responses elicited in mice with nOMVdis were compared to those elicited with nOMVlpxl1 in the presence of hfH. Results demonstrate that the administration of human fH to mice immunized with fHbp containing OMVlpxl1 decreased immunogenicity against fHbp (but not against the OMV as a whole). The majority of the OMV-induced bactericidal immune response (OMVlpxl1 or OMVdis) was versus PorA. Despite a considerable reduction of hfH binding to nOMVdis, and the absence of the vaccine antigen fHbp, immunogenicity in mice was not different from nOMVlpxl1, in the absence or presence of hfH (serum bactericidal titers of 1:64 vs 1:128 after one dose in the nOMVdis and nOMVlpxl1-immunized groups respectively). Therefore, partial inhibition of fH binding did not enhance immunity in this model.
OBJECTIVES: Outer membrane vesicle (OMV) vaccines are used against outbreaks of capsular group B Neisseria meningitidis (MenB) caused by strains expressing particular PorA outer membrane proteins (OMPs). Ferric enterobactin receptor (FetA) is another variable OMP that induces type-specific bactericidal antibodies, and the combination of judiciously chosen PorA and FetA variants in vaccine formulations is a potential approach to broaden protection of such vaccines. METHODS: The OMV vaccine MenPF-1 was generated by genetically modifying N. meningitidis strain 44/76 to constitutively express FetA. Three doses of 25 μg or 50 μg of MenPF-1 were delivered intra-muscularly to 52 healthy adults. RESULTS: MenPF-1 was safe and well tolerated. Immunogenicity was measured by serum bactericidal assay (SBA) against wild-type and isogenic mutant strains. After 3 doses, the proportion of volunteers with SBA titres ≥1:4 (the putative protective titre) was 98% for the wild-type strain, and 77% for the strain 44/76 FetA(on)PorA(off) compared to 51% in the strain 44/76 FetA(off)PorA(off), demonstrating that vaccination with MenPF-1 simultaneously induced FetA and PorA bactericidal antibodies. CONCLUSION: This study provides a proof-of-concept for generating bactericidal antibodies against FetA after OMV vaccination in humans. Prevalence-based choice of PorA and FetA types can be used to formulate a vaccine for broad protection against MenB disease.
Injections with a hypodermic needle and syringe (HNS) are the current standard of care globally, but the use of needles is not without limitation. While a plethora of needle-free injection devices exist, vaccine reformulation is costly and presents a barrier to their widespread clinical application. To provide a simple, needle-free, and broad-spectrum protein antigen delivery platform, we developed novel potassium-doped hydroxyapatite (K-Hap) microparticles with improved protein loading capabilities that can provide sustained local antigen presentation and release. K-Hap showed increased protein adsorption over regular hydroxyapatite (P < 0.001), good structural retention of the model antigen (CRM197) with 1% decrease in α-helix content and no change in β-sheet content upon adsorption, and sustained release in vitro. Needle-free intradermal powder inoculation with K-Hap-CRM197 induced significantly higher IgG1 geometric mean titers (GMTs) than IgG2a GMTs in a BALB/c mouse model (P < 0.001) and induced IgG titer levels that were not different from the current clinical standard (P > 0.05), namely, alum-adsorbed CRM197 by intramuscular (i.m.) delivery. The presented results suggest that K-Hap microparticles may be used as a novel needle-free delivery vehicle for some protein antigens.
Neisseria meningitidis lipopolysaccharide (LPS) has adjuvant properties that can be exploited to assist vaccine immunogenicity. The modified penta-acylated LPS retains the adjuvant properties of hexa-acylated LPS but has a reduced toxicity profile. In this study we investigated whether two modified glycoform structures (LgtE and IcsB) of detoxified penta-acylated LPS exhibited differential adjuvant properties when formulated as native outer membrane vesicles (nOMVs) as compared to the previously described LgtB variant. Detoxified penta-acylated LPS was obtained by disruption of the lpxL1 gene (LpxL1 LPS), and three different glycoforms were obtained by disruption of the lgtB, lgtE or icsB genes respectively. Mice (mus musculus) were immunized with a recombinant PorA P1.7-2,4 (rPorA) protein co-administered with different nOMVs (containing a different PorA serosubtype P1.7,16), each of which expressed one of the three penta-acylated LPS glycoforms. All nOMVs induced IgG responses against the rPorA, but the nOMVs containing the penta-acylated LgtB-LpxL1 LPS glycoform induced significantly greater bactericidal activity compared to the other nOMVs or when the adjuvant was Alhydrogel. Compared to LgtE or IcsB LPS glycoforms, these data support the use of nOMVs containing detoxified, modified LgtB-LpxL1 LPS as a potential adjuvant for future meningococcal protein vaccines.
Introduction: Capsular group B meningococcal disease is a leading cause of childhood meningitis and septicaemia. Up to 10% of sufferers die, and sequelae remain in > 30% of survivors. A vaccine, four component meningococcal group B ( 4CMenB ), designed with the aim to induce broad coverage against this highly variable bacterium, has been licensed in countries including in the European Union, Canada and Australia. Areas covered: Immunogenicity and safety data, published in peer-reviewed literature between 2004 and 2014, are presented in the context of the recent recommendation for the use of the vaccine in infants in the UK. Expert opinion: 4CMenB induces significant reactogenicity when administered with routine infant vaccines, in particular with respect to fever rates. Fevers can be somewhat reduced using paracetamol. The efficacy of the vaccine is unknown but has been extrapolated from effectiveness data obtained from use of one of its components in New Zealand, immunogenicity data from clinical trials and estimation of coverage from in vitro studies. These data suggest that the vaccine will prevent a proportion of invasive meningococcal disease cases in infants and young children. Implementation and well-planned post-marketing surveillance will address uncertainties over field effectiveness.
Staphylococcus aureus remains an important human and animal pathogen. Its pathogenicity is determined in part by expression of the Spa‐immune subversion protein, neutralising the activity of which provides partial protection in murine models, as does experimental infection with live S. aureus with Spa gene deletions followed by antibiotic‐mediated cure in mice. Together, these data raise the question of whether Spa mutant S. aureus might represent a viable vaccine. Here, we find that gamma‐irradiated S. aureus strains, both wild‐type and null mutant of spa, are immunogenic in mice when administered intramuscularly, eliciting large amounts of anti‐S. aureus antibodies, as judged by whole‐cell immunoassay on fixed microorganisms. We used an intravenous challenge system to assess vaccine efficacy, the sensitivity of which was increased by studying renal bacterial concentrations in both kidneys. Despite this, protection from intravenous challenge was not observed (mean difference between vaccinated and unvaccinated mice 0.27 log10 with 95% confidence interval −0.922 to 1.467). Surprisingly, antibody responses elicited against a panel of protective cell surface proteins were very low, indicating that most antibody induced is not protective. Additionally, these data suggest a limited role for irradiated wild‐type or spa mutant S. aureus as vaccines. This is a clear and concise account of a failed Staphylococcus aureus vaccine trial in mice, which contributes to the knowledge in the field. The readers will benefit from a discussion on the mouse strains used in the different vaccine trails, since susceptibility to S. aureus infection is mouse strain‐dependent.
To prevent important infectious diseases such as tuberculosis, malaria and HIV, vaccines inducing greater T cell responses are required. In this study, we investigated whether fusion of the M. tuberculosis antigen 85A to recently described adjuvant IMX313, a hybrid avian C4bp oligomerization domain, could increase T cell responses in pre-clinical vaccine model species. In mice, the fused antigen 85A showed consistent increases in CD4(+) and CD8(+) T cell responses after DNA and MVA vaccination. In rhesus macaques, higher IFN-γ responses were observed in animals vaccinated with MVA-Ag85A IMX313 after both primary and secondary immunizations. In both animal models, fusion to IMX313 induced a quantitative enhancement in the response without altering its quality: multifunctional cytokines were uniformly increased and differentiation into effector and memory T cell subsets was augmented rather than skewed. An extensive in vivo characterization suggests that IMX313 improves the initiation of immune responses as an increase in antigen 85A specific cells was observed as early as day 3 after vaccination. This report demonstrates that antigen multimerization using IMX313 is a simple and effective cross-species method to improve vaccine immunogenicity with potentially broad applicability.
Substantial protection can be provided against the pre-erythrocytic stages of malaria by vaccination first with an adenoviral and then with an modified vaccinia virus Ankara (MVA) poxviral vector encoding the same ME.TRAP transgene. We investigated whether the two vaccine components adenovirus (Ad) and MVA could be coinjected as a mixture to enhance protection against malaria. A single-shot mixture at specific ratios of Ad and MVA (Ad+MVA) enhanced CD8 + T cell-dependant protection of mice against challenge with Plasmodium berghei . Moreover, the degree of protection could be enhanced after homologous boosting with the same Ad+MVA mixture to levels comparable with classic heterologous Ad prime-MVA boost regimes. The mixture increased transgene-specific responses while decreasing the CD8 + T cell antivector immunity compared to each vector used alone, particularly against the MVA backbone. Mixed vector immunization led to increased early circulating interferon-γ (IFN-γ) response levels and altered transcriptional microarray profiles. Furthermore, we found that sequential immunizations with the Ad+MVA mixture led to consistent boosting of the transgene-specific CD8 + response for up to three mixture immunizations, whereas each vector used alone elicited progressively lower responses. Our findings offer the possibility of simplifying the deployment of viral vectors as a single mixture product rather than in heterologous prime-boost regimens.
Accumulating evidence indicates that neutralizing antibodies play an important role in protection from chronic hepatitis C virus (HCV) infection. Efforts to elicit such responses by immunization with intact heterodimeric E1E2 envelope proteins have met with limited success. To determine whether antigenic sites, which are not exposed by the combined E1E2 heterodimer structure, are capable of eliciting neutralizing antibody responses, we expressed and purified each as separate recombinant proteins E1 and E2, from which the immunodominant hypervariable region (HVR-1) was deleted. Immunization of chimpanzees with either E1 or E2 alone induced antigen-specific T-helper cytokines of similar magnitude. Unexpectedly, the capacity to neutralize HCV was observed in E1 but not in animals immunized with E2 devoid of HVR-1. Furthermore, in vivo only E1-vaccinated animals exposed to the heterologous HCV-1b inoculum cleared HCV infection.
Identification of correlates of protection for infectious diseases including malaria is a major challenge and has become one of the main obstacles in developing effective vaccines. We investigated protection against liver-stage malaria conferred by vaccination with adenoviral and Modified Vaccinia Ankara (MVA) vectors expressing pre-erythrocytic malaria antigens. By classifying CD8 + T cells into effector (T E ), effector/memory (T EM ) and central memory (T CM ) subsets using CD62L and CD127 markers, we found striking differences in T cell memory generation. While MVA induced accelerated T CM generation, which could be efficiently boosted by subsequent adenoviral administration, it failed to protect against malaria. In contrast, adenoviral (Ad) vectors, which permit persistent antigen delivery, elicit a prolonged T E and T EM response that requires long intervals for an efficient boost. A preferential T EM phenotype was maintained in liver, blood and spleen following Ad/MVA prime-boost regimens and animals were protected against malaria sporozoite challenge. Blood CD8 + T EM cells correlated with protection against malaria liver-stage infection, assessed by estimation of number of parasites emerging from the liver into the blood. The protective ability of antigen-specific T EM cells was confirmed by transfer experiments into naive recipient mice. Thus, we identify persistent CD8 T EM populations as essential for vaccine-induced pre-erythrocytic protection against malaria, a finding that has important implications for logical vaccine design.
► Viral-vectored vaccines for veterinary and human use: induction of T cell responses. ► Induction of tailored polyfunctional, site-specific and memory immune response. ► Chimpanzee adenoviruses in humans circumvent pre-existing immunity. ► Success of prime-boost regimen in clinical trials for HIV and malaria. ► New technologies for enhancement of the vectors manufacturing and deployment. A little more than a decade after the explosion of research into recombinant live-attenuated or replication-deficient viruses as vaccine platforms, many viral vector-based vaccines have been licensed for animals. Progress has been slower for humans but 2011 will see the licensure of the first viral-vectored vaccine for humans, against Japanese Encephalitis. In addition a vaccine with a viral-vectored component showed efficacy against HIV infection in humans. Viral-based vaccines have an excellent safety profile but must deal with the potential problem of pre-existing anti-vector immunity. Recent successes reflect diverse improvements such as development of new adenovirus serotypes and better prime-boost approaches, suggesting that many viral vectors are approaching their final years as vaccine ‘candidates’ rather than vaccines.
Malaria is a major health problem as nearly half of the human population is exposed to this parasite causing around 600 million clinical cases annually. Prime-boost regimes using simian adenoviral vectors and MVA expressing the clinically relevant Plasmodium falciparum ME.TRAP antigen have shown outstanding protective efficacy in mouse models. We now extend those observations to macaque monkeys. Immunisation with AdCh63 elicited a median response of 869 IFN-γ SFC/million PBMCs to ME.TRAP and responses were boosted by MVA to reach 5256 SFC/million PBMCs, increasing at the same time the breadth of the T cell responses to cover the complete ME.TRAP antigen. Intramuscular vaccination was more immunogenic than the intradermal route, and MVA could be used repeatedly for up to 3 times to boost adenovirus-primed responses. An interval of 16 weeks between repeated MVA injections was optimal to enhance cytokine production by T cells and improve the CD8 multifunctional responses. Antibodies to TRAP were exceptionally high and maintained for a long period of time after the prime-boost regime. These results in non-human primates highlight the potential of this vaccination regime and encourage its future use in clinical trials.
Improving vaccine immunogenicity remains a major challenge in the fight against developing country diseases like malaria and AIDS. We describe a novel strategy to identify new DNA vaccine adjuvants. We have screened components of the Toll-like receptor signalling pathways for their ability to activate pro-inflammatory target genes in transient transfection assays and assessed in vivo adjuvant activity by expressing the activators from the DNA backbone of vaccines. We find that a robust increase in the immune response necessitates co-expression of two activators. Accordingly, the combination of tak1 and tram elicits synergistic reporter activation in transient transfection assays. In a mouse model this combination, but not the individual molecules, induced approximately twofold increases in CD8+ T-cell immune responses. These results indicate that optimal immunogenicity may require activation of distinct innate immune signalling pathways. Thus this strategy offers a novel route to the discovery of a new generation of adjuvants.
The new intradermal DNA delivery technique, termed DNA tattooing might overcome the discrepancy between the encouraging immunogenicity results obtained with DNA vaccines in murine studies and the poor results obtained in non-human primates and humans, the so called “simian barrier”. Here, we demonstrate a 10- to 100-fold increase in the magnitude of vaccine specific T-cell responses in peripheral blood from DNA tattooed rhesus macaques, as compared to T-cell responses in animals immunized via intramuscular (IM) route. A marked increase in the magnitude of the antigen specific T-cell responses as well as an increase in the number of animals responding to the immunogens was observed. These findings in non-human primates suggest that similar results may be observed in humans. Clinical trials are planned to validate tattooing as an optimal method of DNA vaccine delivery in humans.
Broad T cell and B cell responses to multiple HCV antigens are observed early in individuals who control or clear HCV infection. The prevailing hypothesis has been that similar immune responses induced by prophylactic immunization would reduce acute virus replication and protect exposed individuals from chronic infection. Here, we demonstrate that immunization of naïve chimpanzees with a multicomponent HCV vaccine induced robust HCV-specific immune responses, and that all vaccinees exposed to heterologous chimpanzee-adapted HCV 1b J4 significantly reduced viral RNA in serum by 84%, and in liver by 99% as compared to controls (P=0.024 and 0.028, respectively). However, despite control of HCV in plasma and liver in the acute period, in the chronic phase, 3 of 4 vaccinated animals developed persistent infection. Analysis of expression levels of proinflammatory cytokines in serial hepatic biopsies failed to reveal an association with vaccine outcome. However, expression of IDO, CTLA-4 [corrected] and PD-1 levels in liver correlated with clearance or chronicity. Despite early control of virus load, a virus-associated tolerogenic-like state can develop in certain individuals independent of vaccination history.
Indoleamine 2,3-dioxygenase (IDO) is induced by proinflammatory cytokines and by CTLA-4-expressing T cells and constitutes an important mediator of peripheral immune tolerance. In chronic hepatitis C, we found upregulation of IDO expression in the liver and an increased serum kynurenine/tryptophan ratio (a reflection of IDO activity). Huh7 cells supporting hepatitis C virus (HCV) replication expressed higher levels of IDO mRNA than noninfected cells when stimulated with gamma interferon or when cocultured with activated T cells. In infected chimpanzees, hepatic IDO expression decreased in animals that cured the infection, while it remained high in those that progressed to chronicity. For both patients and chimpanzees, hepatic expression of IDO and CTLA-4 correlated directly. Induction of IDO may dampen T-cell reactivity to viral antigens in chronic HCV infection.
Background Preventive and therapeutic vaccine strategies aimed at controlling hepatitis C virus (HCV) infection should mimic the immune responses observed in patients who control or clear HCV, specifically T helper (Th) type 1 and CD8+ cell responses to multiple antigens, including nonstructural protein (NS) 3. Given the experience with human immunodeficiency virus, the best candidates for this are based on DNA prime, pox, or adenovirus boost regimens Methods In rhesus macaques, we compared NS3-expressing DNA prime and adenovirus boost strategy with 2 alternative priming approaches aimed at modifying Th1 and CD8+ responses: DNA adjuvanted with interleukin (IL)–2– and –12–encoding plasmids or Semliki Forest virus (SFV) Results All prime-boost regimens elicited NS3-specific B and T cell responses in rhesus macaques, including CD8+ responses. SFV priming induced higher lymphoproliferation and longer Th1 memory responses. The use of IL-2– and IL-12–expressing vectors resulted in reduced Th2 and antibody responses, which led to increased Th1 skewing but not to an increase in the magnitude of the IFN-γ and CD8+ responses Conclusions All strategies induced Th1 cellular responses to HCV NS3, with fine modulations depending on the different priming approaches. When they are developed for more HCV antigens, these strategies could be beneficial in therapeutic vaccine approaches
The induction of humoral response in ducks by DNA-based immunization against duck hepatitis B virus (DHBV) core protein (DHBc) was investigated. In addition, the amino acid specificity of the induced response was compared by using peptide scanning to that elicited either by protein immunization or during chronic DHBV infection. Immunization of ducks with a plasmid expressing DHBc protein led to the induction of a long-lasting antibody response able to specifically recognize viral protein in chronically infected duck livers. Peptide scanning analysis of anti-DHBc response induced during chronic DHBV infection allowed us to identify six major antigenic regions (AR1 to AR6). The reactivity spectrum of duck sera elicited by protein immunization appeared narrower and was restricted to only four of these antigenic regions in spite of higher anti-DHBc antibody titers. Interestingly, anti-DHBc antibodies induced by DNA-based immunization recognized five of six antigenic regions, and the epitope pattern was broader and more closely related to that observed in chronic viral infections. To gain more insight into the location of antigenic regions, we built a three-dimensional (3-D) model of DHBc protein based on human and duck core sequence alignment data and the HBc 3-D crystal structure. The results suggest that two identified antigenic regions (AR2, amino acids [aa] 64 T-P 84 , and AR5, aa 183 A-R 210 ) are located at positions on the protein surface equivalent to those of the two HBc major epitopes. Moreover, we identified another antigenic region (AR3, aa 99 I-I 112 ) that was recognized by all sera from chronically infected, DNA- or protein-immunized ducks within the large 45-aa insertion in DHBc protein, suggesting that this region, which lacks HBc, is externally exposed.
Prophylactic hepatitis C virus (HCV) vaccine trials with human volunteers are pending. There is an important need for immunological end points which correlate with vaccine efficacy and which do not involve invasive procedures, such as liver biopsies. By using a multicomponent DNA priming-protein boosting vaccine strategy, naïve chimpanzees were immunized against HCV structural proteins (core, E1, and E2) as well as a nonstructural (NS3) protein. Following immunization, exposure to the heterologous HCV 1b J4 subtype resulted in a peak of plasma viremia which was lower in both immunized animals. Compared to the naïve infection control and nine additional historical controls which became chronic, vaccinee 2 (Vac2) rapidly resolved the infection, while the other (Vac1) clearly controlled HCV infection. Immunization induced antibodies, peptide-specific gamma interferon (IFN-γ), protein-specific lymphoproliferative responses, IFN-γ, interleukin-2 (IL-2), and IL-4 T-helper responses in both vaccinees. However, the specificities were markedly different: Vac2 developed responses which were lower in magnitude than those of Vac1 but which were biased towards Th1-type cytokine responses for E1 and NS3. This proof-of-principle study in chimpanzees revealed that immunization with a combination of nonstructural and structural antigens elicited T-cell responses associated with an alteration of the course of infection. Our findings provide data to support the concept that the quality of the response to conserved epitopes and the specific nature of the peripheral T-helper immune response are likely pivotal factors influencing the control and clearance of HCV infection.
Chronic hepatitis C virus (HCV) infection in humans is associated with an impairment of dendritic cells (DC). It has been hypothesized that impairment of DC function may be a central mechanism facilitating the establishment of a chronic carrier state. However, the majority of patients studied with DC impairment to date have been identified and, thus, inadvertently selected because of clinical manifestations leading to their diagnosis, which may have been many years following actual infection. We set out to determine whether impaired DC function occurred in the earlier asymptomatic phase of infection and turned to a well-defined cohort of HCV-infected chimpanzees in which the specific date of infection and the nature of the inoculum were well characterized. Results revealed that, in contrast to the observations in human subjects with advanced clinical hepatitis, there was neither impairment of the allostimulatory capacity of monocyte-derived DC from HCV chronic carriers nor impairment of the maturation process. Decreased allostimulatory capacity was only detected in 2 animals and, interestingly, in those that possessed the highest viral loads. Nevertheless, HCV sequences were undetectable in any of the DC derived from HCV-infected chimpanzees. In conclusion, these findings suggest that the mechanisms of establishing persistent HCV infection are separate and independent from those responsible for impaired DC function. Indeed, the maturation and allostimulatory impairment, as described in patient studies, are not necessary prerequisites but rather possible consequences of persistent and active HCV infection associated with disease progression. (H epatology 2003;38:851-858.)
DNA vaccination may represent an interesting strategy for early life immunization. However, in some cases, this approach has been shown to induce a tolerance rather than immunity. We have compared the efficiency of neonatal DNA or protein immunization against hepadnavirus envelope protein using the duck hepatitis B virus (DHBV) model. Three-day-old ducklings were immunized with either a plasmid encoding the DHBV pre-S/S large envelope protein (L), or a recombinant preS protein, followed by sequential DNA or protein boosts at weeks 4 and 15. Our results showed that genetic immunization of duck neonates induced specific humoral response to DHBV L protein. Interestingly, an enhanced antibody response was elicited when animals received DNA priming–DNA boosting as compared to DNA priming–protein boosting.
The outcome and protective efficacy of maternal antibodies elicited by DNA immunization to the large (L) hepadnavirus envelope protein were studied using the duck hepatitis B virus (DHBV) model. Following genetic immunization of breeding ducks with a DHBV L protein gene-bearing plasmid, specific and highly neutralizing antibodies were transferred from the sera of immunized ducks, via the egg yolk, to the progeny of vaccinees. Interestingly, large amounts (60 to 100 mg/egg) of high-titer and L protein-specific yolk immunoglobulins (immunoglobulin Y) accumulated in the egg yolk. These results suggest that eggs from genetically immunized avians may represent a potent source of DNA-designed antibodies specific to viral antigen. Importantly, these antibodies are vertically transmitted and protect offspring against high-titer DHBV challenge.
To date, no detailed analysis of the neutralization properties of duck hepatitis B virus (DHBV) has been reported, and it is not clear whether any of the known neutralization epitopes correspond to the viral receptor binding site or to sequences involved in the cell entry pathway. We demonstrate here that antibodies directed against two overlapping peptides (amino acids 83 to 97 and 93 to 107), covering the sequences of most DHBV pre-S neutralizing epitopes, both inhibit virus binding to primary duck hepatocytes and neutralize virus infectivity. An extensive mutagenesis of the motif 88 WTP 90 , which is the shortest sequence of the epitope recognized by the virus-neutralizing monoclonal antibody (MAb) 900 was performed in order to define the amino acids involved in these interactions. Single point mutations within this epitope affected neither virus replication nor infectivity but abolished virus neutralization by MAb 900 completely. Interestingly, mutants with two and three consecutive residue replacements (SIP and SIH) within this epitope retained replication competence but were no longer infectious. The loss of infectivity of SIH and SIP mutant particles was associated with significantly reduced binding to primary duck hepatocytes and could be rescued by trans complementation with wild-type pre-S protein. Taken together, these results indicate that each amino acid of the DHBV pre-S sequence 88 WTP 90 is critical for recognition by the neutralizing MAb 900 and that replacement of the first two or all three residues strongly reduces virus interaction with hepatocytes and abrogates infectivity. These data imply that the motif 88 WTP 90 contains key residues which are critical for interaction with both the neutralizing MAb and the host cell.
Background Zika virus (ZIKV) has become a global threat with immediate need for accurate diagnostics, efficacious vaccines and therapeutics. Several ZIKV envelope (Env)-based vaccines have been developed recently. However, many commercially available ZIKV Env are based on the African lineage and produced in insect cells. Here, we sought to produce Asian-lineage ZIKV Env in mammalian cells for research and clinical applications. Methods We designed various gene expression constructs to optimize the production of ZIKV using prM-Env and full or C-terminal truncations of Env; with or without a rat CD4 fusion partner to allow large-scale production of soluble protein in mammalian HEK293 cells. Protein expression was verified by mass spectrometry and western-blot with a pan-flavivirus antibody, a ZIKV Env monoclonal antibody and with immune sera from adenoviral (ChAdOx1) ZIKV Env-vaccinated mice. The resulting Env-CD4 was used as a coating reagent for immunoassay (ELISA) using both mouse and human seropositive sera. Results Replacement of the C-terminus transmembrane Env domain by a rat CD4 and addition of prM supported optimal expression and secretion of Env. Binding between the antigens and the antibodies was similar to binding when using commercially available ZIKV Env reagents. Furthermore, antibodies from ZIKV patients bound ZIKV Env-CD4 in ELISA assays, whereas sera from healthy blood donors yielded minimal OD background. The serological outcomes of this assay correlated also with ZIKV neutralisation capacity in vitro. Conclusions Results obtained from this study indicate the potential of the Asian-lineage Zika Env-CD4 and Env proteins in ELISA assays to monitor humoral immune responses in upcoming clinical trials as well as a sero-diagnostic tool in ZIKV infection.
Outer membrane vesicle (OMV)- based vaccines have been used to provide strain-specific protection against capsular group B Neisseria meningitidis infections, but the full breadth of the immune response against the components of the OMV has not been established. Sera from adults vaccinated with an OMV vaccine were used to screen 91 outer membrane proteins (OMPs) incorporated in an antigen microarray panel. Antigen-specific IgG levels were quantified pre-vaccination, and after 12 and 18 weeks. These results were compared with IgG levels from mice vaccinated with the same OMV vaccine. The repertoires of highly responding antigens in humans and mice overlapped, but were not identical. The highest responding antigens to human IgG comprised four integral OMPs (PorA, PorB, OpcA and PilQ), a protein which promotes the stability of PorA and PorB (RmpM) and two lipoproteins (BamC and GNA1162). These observations will assist in evaluating the role of minor antigen components within OMVs in providing protection against meningococcal infection. In addition, the relative dominance of responses to integral OMPs in humans emphasizes the importance of this subclass and points to the value of maintaining conformational epitopes from integral membrane proteins in vaccine formulations.
Background & Aims: Studies in the murine model suggest that injection of DNA encoding hepatitis B virus structural proteins is promising for the induction of a specific immune response. We used the duck hepatitis B virus (DHBV) model to study the protective and therapeutic effects of naked DNA immunization against hepadnaviral large envelope protein. Methods: A pCI-preS/S plasmid expressing the DHBV large protein was used for intramuscular immunization of ducks. The humoral response was tested by enzyme-linked immunosorbent assay, immunoblotting, neutralization, and in vivo protection tests. For DNA therapy, DHBV-carrier ducks received four injections of this plasmid. Viremia was monitored for 10 months; thereafter, liver biopsies were performed. Results: Immunization with pCI-preS/S plasmid induced a specific, long-lasting, neutralizing, and highly protective anti-preS humoral response in uninfected animals. After pCI-preS/S treatment, a significant and sustained decrease in serum and liver DHBV DNA was observed for carrier ducks compared with the controls. Conclusions: DNA immunization against DHBV large protein results in a potent and protective anti-preS response in the duck model. The results of long-term follow-up of DNA-treated chronically infected ducks are promising and show the usefulness of this model for the study of genetic immunization in chronic hepatitis B therapy.