26 results found
Martin GM, Russell RA, Mundsperger P, et al., 2023, Profound structural conservation of chemically cross-linked HIV-1 envelope glycoprotein experimental vaccine antigens., npj Vaccines, Vol: 8, ISSN: 2059-0105
Chemical cross-linking is used to stabilize protein structures with additional benefits of pathogen and toxin inactivation for vaccine use, but its use has been restricted by the potential for local or global structural distortion. This is of particular importance when the protein in question requires a high degree of structural conservation for inducing a biological outcome such as the elicitation of antibodies to conformationally sensitive epitopes. The HIV-1 envelope glycoprotein (Env) trimer is metastable and shifts between different conformational states, complicating its use as a vaccine antigen. Here we have used the hetero-bifunctional zero-length reagent 1-Ethyl-3-(3-Dimethylaminopropyl)-Carbodiimide (EDC) to cross-link two soluble Env trimers, selected well-folded trimer species using antibody affinity, and transferred this process to good manufacturing practice (GMP) for experimental medicine use. Cross-linking enhanced trimer stability to biophysical and enzyme attack. Cryo-EM analysis revealed that cross-linking retained the overall structure with root-mean-square deviations (RMSDs) between unmodified and cross-linked Env trimers of 0.4-0.5 Å. Despite this negligible distortion of global trimer structure, we identified individual inter-subunit, intra-subunit, and intra-protomer cross-links. Antigenicity and immunogenicity of the trimers were selectively modified by cross-linking, with cross-linked ConS retaining bnAb binding more consistently than ConM. Thus, the EDC cross-linking process improves trimer stability whilst maintaining protein folding, and is readily transferred to GMP, consistent with the more general use of this approach in protein-based vaccine design.
Szubert AJ, Pollock KM, Cheeseman HM, et al., 2023, COVAC1 phase 2a expanded safety and immunogenicity study of a self-amplifying RNA vaccine against SARS-CoV-2., EClinicalMedicine, Vol: 56, Pages: 1-13, ISSN: 2589-5370
BACKGROUND: Lipid nanoparticle (LNP) encapsulated self-amplifying RNA (saRNA) is well tolerated and immunogenic in SARS-CoV-2 seronegative and seropositive individuals aged 18-75. METHODS: A phase 2a expanded safety and immunogenicity study of a saRNA SARS-CoV-2 vaccine candidate LNP-nCoVsaRNA, was conducted at participating centres in the UK between 10th August 2020 and 30th July 2021. Participants received 1 μg then 10 μg of LNP-nCoVsaRNA, ∼14 weeks apart. Solicited adverse events (AEs) were collected for one week post-each vaccine, and unsolicited AEs throughout. Binding and neutralisating anti-SARS-CoV-2 antibody raised in participant sera was measured by means of an anti-Spike (S) IgG ELISA, and SARS-CoV-2 pseudoneutralisation assay. (The trial is registered: ISRCTN17072692, EudraCT 2020-001646-20). FINDINGS: 216 healthy individuals (median age 51 years) received 1.0 μg followed by 10.0 μg of the vaccine. 28/216 participants were either known to have previous SARS-CoV2 infection and/or were positive for anti-Spike (S) IgG at baseline. Reactogenicity was as expected based on the reactions following licensed COVID-19 vaccines, and there were no serious AEs related to vaccination. 80% of baseline SARS-CoV-2 naïve individuals (147/183) seroconverted two weeks post second immunization, irrespective of age (18-75); 56% (102/183) had detectable neutralising antibodies. Almost all (28/31) SARS-CoV-2 positive individuals had increased S IgG binding antibodies following their first 1.0 μg dose with a ≥0.5log10 increase in 71% (22/31). INTERPRETATION: Encapsulated saRNA was well tolerated and immunogenic in adults aged 18-75 years. Seroconversion rates in antigen naïve were higher than those reported in our dose-ranging study. Further work is required to determine if this difference is related to a longer dosing interval (14 vs. 4 weeks) or dosing with 1.0 μg followed by 10.0 μg. Boosting of S IgG an
Day S, Kaur C, Cheeseman H, et al., 2022, Comparison of blood and lymph node cells after intramuscular injection with HIV envelope immunogens, Frontiers in Immunology, Vol: 13, Pages: 1-17, ISSN: 1664-3224
Background:Harnessing CD4+ T cell help in the lymph nodes through rational antigen design could enhance formation of broadly neutralising antibodies (bNAbs) during experimental HIV immunisation. This process has remained hidden due to difficulty with direct study, with clinical studies instead focusing on responses in the blood as a proxy for the secondary lymphoid tissue. Methods:To address this, lymph node cells (LNC) were collected using ultrasound guided fine needle aspiration of axillary lymph nodes from 11 HIV negative participants in an experimental HIV immunogen study (European AIDS Vaccine Initiative EAVI2020_01 study, NCT04046978). Cells from lymph node and blood (PBMC), were collected after intramuscular injection with HIV Env Mosaic immunogens based on HIV Envelope glycoprotein and combined with a liposomal toll-like receptor-4 adjuvant; monophosphoryl lipid A. Simultaneously sampled cells from both blood and lymph node in the same donors were compared for phenotype, function, and antigen-specificity. Results:Unsupervised cluster analysis revealed tissue-specific differences in abundance, distribution, and functional response of LNC compared with PBMC. Monocytes were virtually absent from LNC, which were significantly enriched for CD4+ T cells compared with CD8+ T cells. T follicular helper cells with germinal centre features were enriched in LNC, which contained specific CD4+ and CD8+ T cell subsets including CD4+ T cells that responded after a single injection with HIV Env Mosaic immunogens combined with adjuvant. Tissue-specific differences in response to an MHC-II dependent superantigen, staphylococcal enterotoxin B, indicated divergence in antigen presentation function between blood and lymph node. Conclusions:LNC are phenotypically and functionally distinct from PBMC, suggesting that whole blood is only a limited proxy of the T cell lymphatic response to immunisation. HIV-specific CD4+ T cells in the lymph node are rapidly inducible upon experimen
Elliott T, Cheeseman HM, Evans AB, et al., 2022, Enhanced immune responses following heterologous vaccination with self-amplifying RNA and mRNA COVID-19 vaccines, PLoS Pathogens, Vol: 18, Pages: 1-20, ISSN: 1553-7366
The optimal vaccination strategy to boost responses in the context of pre-existing immune memory to the SARS-CoV-2 spike (S) glycoprotein is an important question for global public health. To address this, we explored the SARS-CoV-2-specific humoral and cellular immune responses to a novel self-amplifying RNA (saRNA) vaccine followed by a UK authorised mRNA vaccine (BNT162b2) in individuals with and without previous COVID-19, and compared these responses with those who received an authorised vaccine alone. 35 subjects receiving saRNA (saRNA group) as part of the COVAC1 clinical trial and an additional 40 participants receiving an authorised SARS-CoV-2 vaccine only (non-saRNA group) were recruited. Antibody responses were measured by ELISA and a pseudoneutralisation assay for wildtype, Delta and Omicron variants. Cellular responses were measured by IFN-ƴ ELISpot and an activation induced marker (AIM) assay. Approximately 50% in each group had previous COVID-19 prior to vaccination, confirmed by PCR or antibody positivity on ELISA. All of those who received saRNA subsequently received a full course of an authorised vaccine. The majority (83%) of those receiving saRNA who were COVID-19 naïve at baseline seroconverted following the second dose, and those with previous COVID-19 had an increase in antibody titres two weeks following saRNA vaccination (median 27-fold), however titres were lower when compared to mRNA vaccination. Two weeks following the 2nd authorised mRNA vaccine dose, binding and neutralising antibody titres were significantly higher in the saRNA participants with previous COVID-19, compared to non-saRNA, or COVID-19 naive saRNA participants. Cellular responses were again highest in this group, with a higher proportion of spike specific CD8+ than CD4+ T cells when compared to those receiving the mRNA vaccine only. These findings suggest an immunological benefit of increased antigen exposure, both from natural infection and vaccination, particularly e
Pollock KM, Cheeseman HM, Szubert AJ, et al., 2022, Safety and immunogenicity of a self-amplifying RNA vaccine against COVID-19: COVAC1, a phase I, dose-ranging trial, EClinicalMedicine, Vol: 44, ISSN: 2589-5370
Background: Lipid nanoparticle (LNP) encapsulated self-amplifying RNA (saRNA) is a novel technology formulated as a low dose vaccine against COVID-19. Methods: A phase I first-in-human dose-ranging trial of a saRNA COVID-19 vaccine candidate LNP-nCoVsaRNA, was conducted at Imperial Clinical Research Facility, and participating centres in London, UK, between 19th June to 28th October 2020. Participants received two intramuscular (IM) injections of LNP-nCoVsaRNA at six different dose levels, 0.1-10.0μg, given four weeks apart. An open-label dose escalation was followed by a dose evaluation. Solicited adverse events (AEs) were collected for one week from enrolment, with follow-up at regular intervals (1-8 weeks). The binding and neutralisation capacity of anti-SARS-CoV-2 antibody raised in participant sera was measured by means of an anti-Spike (S) IgG ELISA, immunoblot, SARS-CoV-2 pseudoneutralisation and wild type neutralisation assays. (The trial is registered: ISRCTN17072692, EudraCT 2020-001646-20). Findings: 192 healthy individuals with no history or serological evidence of COVID-19, aged 18-45 years were enrolled. The vaccine was well tolerated with no serious adverse events related to vaccination. Seroconversion at week six whether measured by ELISA or immunoblot was related to dose (both p<0.001), ranging from 8% (3/39; 0.1μg) to 61% (14/23; 10.0μg) in ELISA and 46% (18/39; 0.3μg) to 87% (20/23; 5.0μg and 10.0μg) in a post-hoc immunoblot assay. Geometric mean (GM) anti-S IgG concentrations ranged from 74 (95% CI, 45-119) at 0.1μg to 1023 (468-2236) ng/mL at 5.0μg (p<0.001) and was not higher at 10.0μg. Neutralisation of SARS-CoV-2 by participant sera was measurable in 15% (6/39; 0.1μg) to 48% (11/23; 5.0μg) depending on dose level received. Interpretation: Encapsulated saRNA is safe for clinical development, is immunogenic at low dose levels but failed to induce 100% seroconversion. Modifications to optimis
Cole ME, Guo Y, Cheeseman HM, et al., 2022, Multicolor Flow Cytometry and High-Dimensional Data Analysis to Probe Complex Questions in Vaccinology., Methods Mol Biol, Vol: 2414, Pages: 433-447
Vaccines induce a highly complex immune reaction in secondary lymphoid organs to generate immunological memory against an antigen or antigens of interest. Measurement of post immunization immune responses generated by specialized lymphocyte subsets requires time-dependent sampling, usually of the blood. Several T and B cell subsets are involved in the reaction, including CD4 and CD8 T cells, T follicular helper cells (Tfh), and germinal center B cells alongside their circulating (c) counterparts; cTfh and antibody secreting cells. Multicolor flow cytometry of peripheral blood mononuclear cells (PBMC) coupled with high-dimensional analysis offers an opportunity to study these cells in detail. Here we demonstrate a method by which such data can be generated and analysed using software that renders multidimensional data on a two dimensional map to identify rare vaccine-induced T and B cell subsets.
Moshe M, Daunt A, Flower B, et al., 2021, SARS-CoV-2 lateral flow assays for possible use in national covid-19 seroprevalence surveys (REACT2): diagnostic accuracy study, BMJ: British Medical Journal, Vol: 372, Pages: 1-8, ISSN: 0959-535X
Objective: To evaluate the performance of new lateral flow immunoassays (LFIAs) suitable for use in a national COVID-19 seroprevalence programme (REACT2).Design: Laboratory sensitivity and specificity analyses were performed for seven LFIAs on a minimum of 200 sera from individuals with confirmed SARS-CoV-2 infection, and 500 pre-pandemic sera respectively. Three LFIAs were found to have a laboratory sensitivity superior to the finger-prick sensitivity of the LFIA currently used in REACT2 seroprevalence studies (84%). These LFIAs were then further evaluated through finger-prick testing on participants with confirmed previous SARS-CoV-2 infection. Two LFIAs (Surescreen, Panbio) were evaluated in clinics in June-July, 2020, and a third LFIA (AbC-19) in September, 2020. A Spike protein enzyme-linked immunoassay (S-ELISA) and hybrid double antigen binding assay (DABA) were used as laboratory reference standards.Setting: Laboratory analyses were performed at Imperial College, London and University facilities in London, UK. Research clinics for finger-prick sampling were run in two affiliated NHS trusts.Participants: Sensitivity analysis on sera were performed on 320 stored samples from previous participants in the REACT2 programme with confirmed previous SARS-CoV-2 infection. Specificity analysis was performed using 1000 pre-pandemic sera. 100 new participants with confirmed previous SARS-CoV-2 infection attended study clinics for finger-prick testing.Main outcome measures: The accuracy of LFIAs in detecting IgG antibodies to SARS-CoV-2 in comparison to two in-house ELISAs.Results: The sensitivity of seven new LFIAs using sera varied between 69% and 100% (vs S-ELISA/hybrid DABA). Specificity using sera varied between 99.6% and 100%. Sensitivity on finger-prick testing for Panbio, Surescreen and AbC-19 was 77% (CI 61.4 to 88.2), 86% (CI 72.7 to 94.8) and 69% (CI 53.8 to 81.3) respectively vs S-ELISA/hybrid DABA. Sensitivity for sera from matched clinical samples performe
Klein K, Nankya I, Nickel G, et al., 2021, Deep gene sequence cluster analyses of multi-virus-infected mucosal tissue reveal enhanced transmission of acute HIV-1, Journal of Virology, Vol: 95, Pages: 1-17, ISSN: 0022-538X
Exposure of the genital mucosa to a genetically diverse viral swarm from the donor HIV-1 can result in breakthrough and systemic infection by a single transmitted/founder (TF) virus in the recipient. The highly diverse HIV-1 envelope (Env) in this inoculating viral swarm may have a critical role in transmission and subsequent immune response. Thus, chronic (Envchronic) and acute (Envacute) Env chimeric HIV-1 were tested using multivirus competition assays in human mucosal penile and cervical tissues. Viral competition analysis revealed that Envchronic viruses resided and replicated mainly in the tissue, while Envacute viruses penetrated the human tissue and established infection of CD4+ T cells more efficiently. Analysis of the replication fitness, as tested in peripheral blood mononuclear cells (PBMCs), showed similar replication fitness of Envacute and Envchronic viruses, which did not correlate with transmission fitness in penile tissue. Further, we observed that chimeric Env viruses with higher replication in genital mucosal tissue (chronic Env viruses) had higher binding affinity to C-type lectins. Data presented herein suggest that the inoculating HIV-1 may be sequestered in the genital mucosal tissue (represented by chronic Env HIV-1) but that a single HIV-1 clone (e.g., acute Env HIV-1) can escape this trapped replication for systemic infection.
Since the emergence of COVID-19, caused by the SARS-CoV-2 virus, at the end of 2019 there has been an explosion of vaccine development. By the 1st September 2020, a staggering number of vaccines (over 200) had started pre-clinical development of which 39 had entered clinical trials, including some approaches that have not previously been licensed for human vaccines. Vaccines have been widely considered as part of the exit strategy to enable the return to previous patterns of working, schooling and socialising. Importantly, to effectively control the COVID-19 pandemic, production needs to be scaled up from a small number of pre-clinical doses to enough filled vials to immunise the world's population, which requires close engagement with manufacturers and regulators. It will require a global effort to control the virus, necessitating equitable access for all countries to effective vaccines. This review explores the immune responses required to protect against SARS-CoV-2 and the potential for vaccine-induced immunopathology. It describes the profile of the different platforms and the advantages and disadvantages of each approach. The review also addresses the critical steps between promising pre-clinical leads and manufacturing at scale. The issues faced during this pandemic and the platforms being developed to address it will be invaluable for future outbreak control. Nine months after the outbreak began, we are at a point where pre-clinical and early clinical data is being generated for the vaccines, an overview of this important area will help our understanding of the next phases.
Flower B, Brown JC, Simmons B, et al., 2020, Clinical and laboratory evaluation of SARS-CoV-2 lateral flow assays for use in a national COVID-19 sero-prevalence survey, Thorax, Vol: 75, Pages: 1082-1088, ISSN: 0040-6376
BackgroundAccurate antibody tests are essential to monitor the SARS-CoV-2 pandemic. Lateral flow immunoassays (LFIAs) can deliver testing at scale. However, reported performance varies, and sensitivity analyses have generally been conducted on serum from hospitalised patients. For use in community testing, evaluation of finger-prick self-tests, in non-hospitalised individuals, is required.MethodsSensitivity analysis was conducted on 276 non-hospitalised participants. All had tested positive for SARS-CoV-2 by RT-PCR and were ≥21d from symptom-onset. In phase I we evaluated five LFIAs in clinic (with finger-prick) and laboratory (with blood and sera) in comparison to a) PCR-confirmed infection and b) presence of SARS-CoV-2 antibodies on two “in-house” ELISAs. Specificity analysis was performed on 500 pre-pandemic sera. In phase II, six additional LFIAs were assessed with serum.Findings95% (95%CI [92.2, 97.3]) of the infected cohort had detectable antibodies on at least one ELISA. LFIA sensitivity was variable, but significantly inferior to ELISA in 8/11 assessed. Of LFIAs assessed in both clinic and laboratory, finger-prick self-test sensitivity varied from 21%-92% vs PCR-confirmed cases and 22%-96% vs composite ELISA positives. Concordance between finger-prick and serum testing was at best moderate (kappa 0.56) and, at worst, slight (kappa 0.13). All LFIAs had high specificity (97.2% - 99.8%).InterpretationLFIA sensitivity and sample concordance is variable, highlighting the importance of evaluations in setting of intended use. This rigorous approach to LFIA evaluation identified a test with high specificity (98.6% (95%CI [97.1, 99.4])), moderate sensitivity (84.4% with fingerprick (95%CI [70.5, 93.5])), and moderate concordance, suitable for seroprevalence surveys.
Abraham S, Juel HB, Bang P, et al., 2019, Safety and immunogenicity of the chlamydia vaccine candidate CTH522 adjuvanted with CAF01 liposomes or aluminium hydroxide: a first-in-human, randomised, double-blind, placebo-controlled, phase 1 trial, Lancet Infectious Diseases, Vol: 19, Pages: 1091-1100, ISSN: 1473-3099
BACKGROUND: Chlamydia is the most common sexually transmitted bacterial infection worldwide. National screening programmes and antibiotic treatment have failed to decrease incidence, and to date no vaccines against genital chlamydia have been tested in clinical trials. We aimed to assess the safety and immunogenicity, in humans, of a novel chlamydia vaccine based on a recombinant protein subunit (CTH522) in a prime-boost immunisation schedule. METHODS: This phase 1, first-in-human, double-blind, parallel, randomised, placebo-controlled trial was done at Hammersmith Hospital in London, UK, in healthy women aged 19-45 years. Participants were randomly assigned (3:3:1) to three groups: CTH522 adjuvanted with CAF01 liposomes (CTH522:CAF01), CTH522 adjuvanted with aluminium hydroxide (CTH522:AH), or placebo (saline). Participants received three intramuscular injections of 85 μg vaccine (with adjuvant) or placebo to the deltoid region of the arm at 0, 1, and 4 months, followed by two intranasal administrations of 30 μg unadjuvanted vaccine or placebo (one in each nostril) at months 4·5 and 5·0. The primary outcome was safety and the secondary outcome was humoral immunogenicity (anti-CTH522 IgG seroconversion). This study is registered with Clinicaltrials.gov, number NCT02787109. FINDINGS: Between Aug 15, 2016, and Feb 13, 2017, 35 women were randomly assigned (15 to CTH522:CAF01, 15 to CTH522:AH, and five to placebo). 32 (91%) received all five vaccinations and all participants were included in the intention-to-treat analyses. No related serious adverse reactions were reported, and the most frequent adverse events were mild local injection-site reactions, which were reported in all (15 [100%] of 15) participants in the two vaccine groups and in three (60%) of five participants in the placebo group (p=0·0526 for both comparisons). Intranasal vaccination was not associated with a higher frequency of related local reactions (reported in seven [47%]
Cheeseman HM, Day S, McFarlane LR, et al., 2018, Combined Skin and Muscle DNA Priming Provides Enhanced Humoral Responses to a Human Immunodeficency Virus Type 1 Clade C Envelope Vaccine, Human Gene Therapy, Vol: 29, Pages: 1011-1028, ISSN: 1043-0342
© Copyright 2018, Mary Ann Liebert, Inc., publishers2018. Intradermal (i.d.) and intramuscular (i.m.) injections when administered with or without electroporation (EP) have the potential to tailor the immune response to DNA vaccination. This Phase I randomized controlled clinical trial in human immunodeficiency virus type 1-negative volunteers investigated whether the site and mode of DNA vaccination influences the quality of induced cellular and humoral immune responses following the DNA priming phase and subsequent protein boost with recombinant clade C CN54 gp140. A strategy of concurrent i.d. and i.m. DNA immunizations administered with or without EP was adopted. Subtle differences were observed in the shaping of vaccine-induced virus-specific CD4+ and CD8+ T cell-mediated immune responses between groups receiving: i.d.EP+ i.m., i.d. + i.m.EP, and i.d.EP+ i.m.EPregimens. The DNA priming phase induced 100% seroconversion in all of the groups. A single, non-adjuvanted protein boost induced a rapid and profound increase in binding antibodies in all groups, with a trend for higher responses in i.d.EP+ i.m.EP. The magnitude of antigen-specific binding immunoglobulin G correlated with neutralization of closely matched clade C 93MW965 virus and Fc-dimer receptor binding (FcγRIIa and FcγRIIIa). These results offer new perspectives on the use of combined skin and muscle DNA immunization in priming humoral and cellular responses to recombinant protein.
Kinnear E, Lambert L, McDonald JU, et al., 2017, Corrigendum: Airway T cells protect against RSV infection in the absence of antibody, Mucosal Immunology, Vol: 11, Pages: 290-290, ISSN: 1933-0219
Correction to:Mucosal Immunology(2018)11,249–256; doi:10.1038/mi.2017.46; published online 24 May 2017
Kinnear E, Lambert L, McDonald JU, et al., 2017, Airway T cells protect against RSV infection in the absence of antibody, Mucosal Immunology, Vol: 11, Pages: 249-256, ISSN: 1933-0219
Tissue resident memory T (Trm) cells act as sentinels and early responders to infection. Respiratory syncytial virus (RSV)-specific Trm cells have been detected in the lungs after human RSV infection, but whether they have a protective role is unknown. To dissect the protective function of Trm cells, BALB/c mice were infected with RSV; infected mice developed antigen-specific CD8(+) Trm cells (CD103(+)/CD69(+)) in the lungs and airways. Intranasally transferring cells from the airways of previously infected animals to naïve animals reduced weight loss on infection in the recipient mice. Transfer of airway CD8 cells led to reduced disease and viral load and increased interferon-γ in the airways of recipient mice, while CD4 transfer reduced tumor necrosis factor-α in the airways. Because DNA vaccines induce a systemic T-cell response, we compared vaccination with infection for the effect of memory CD8 cells generated in different compartments. Intramuscular DNA immunization induced RSV-specific CD8 T cells, but they were immunopathogenic and not protective. Notably, there was a marked difference in the induction of Trm cells; infection but not immunization induced antigen-specific Trm cells in a range of tissues. These findings demonstrate a protective role for airway CD8 against RSV and support the need for vaccines to induce antigen-specific airway cells.Mucosal Immunology advance online publication, 24 May 2017; doi:10.1038/mi.2017.46.
Arakelyan A, Fitzgerald W, King DF, et al., 2017, Flow virometry analysis of envelope glycoprotein conformations on individual HIV virions, Scientific Reports, Vol: 7, ISSN: 2045-2322
HIV-1 envelope proteins (Envs) play a critical role in HIV infection. In a correct trimeric conformation, Env mediates virus–cell binding and fusion. Malfunctioning of this machinery renders virions incapable of infecting cells. Each HIV-1 virion carries 10–14 Envs, and therefore a defective Env may not necessarily render a HIV virion non-infectious, since other Env on the same virion may still be functional. Alternatively, it is possible that on a given virion either all the spikes are defective or all are functional. Here, we investigate Env conformations on individual virions using our new nanotechnology, “flow virometry”, and a panel of antibodies that discriminate between various Env conformations. We found that the majority of HIV-1 virions carry either only trimeric (“functional”) or only defective spikes. The relatively small subfraction of virions that carry both functional and nonfunctional Envs contributes little to HIV infection of human lymphoid tissue ex vivo. The observation that the majority of virions exclusively express either functional or nonfunctional forms of Env has important implications for understanding the role of neutralizing and non-neutralizing antibodies in the immune control of HIV infection as well as for the development of effective prophylactic strategies.
Cheeseman HM, Olejniczak NJ, Rogers PM, et al., 2016, Broadly neutralizing antibodies display potential for prevention of HIV-1 infection of mucosal tissue superior to that of nonneutralizing antibodies, Journal of Virology, Vol: 91, ISSN: 1098-5514
Definition of the key parameters mediating effective antibody blocking of HIV-1 acquisition within mucosal tissue may prove critical to effective vaccine development and the prophylactic use of monoclonal antibodies. Although direct antibody-mediated neutralization is highly effective against cell-free virus, antibodies targeting different sites of envelope vulnerability may display differential activity against mucosal infection. Nonneutralizing antibodies (nnAbs) may also impact mucosal transmission events through Fc-gamma receptor (FcγR)-mediated inhibition. In this study, a panel of broadly neutralizing antibodies (bnAbs) and nnAbs, including those associated with protection in the RV144 vaccine trial, were screened for the ability to block HIV-1 acquisition and replication across a range of cellular and mucosal tissue models. Neutralization potency, as determined by the TZM-bl infection assay, did not fully predict activity in mucosal tissue. CD4-binding site (CD4bs)-specific bnAbs, in particular VRC01, were consistent in blocking HIV-1 infection across all cellular and tissue models. Membrane-proximal external region (MPER) (2F5) and outer domain glycan (2G12) bnAbs were also efficient in preventing infection of mucosal tissues, while the protective efficacy of bnAbs targeting V1-V2 glycans (PG9 and PG16) was more variable. In contrast, nnAbs alone and in combinations, while active in a range of cellular assays, were poorly protective against HIV-1 infection of mucosal tissues. These data suggest that tissue resident effector cell numbers and low FcγR expression may limit the potential of nnAbs to prevent establishment of the initial foci of infection. The solid protection provided by specific bnAbs clearly demonstrates their superior potential over that of nonneutralizing antibodies for preventing HIV-1 infection at the mucosal portals of infection.IMPORTANCE Key parameters mediating effective antibody blocking of HIV-1 acquisition within mucosal
Cheeseman HM, Carias AM, Evans AB, et al., 2016, Expression profile of human Fc receptors in mucosal tissue: implications for antibody-dependent cellular effector functions targeting HIV-1 transmission, PLOS One, Vol: 11, ISSN: 1932-6203
The majority of new Human Immunodeficiency Virus (HIV)-1 infections are acquired via sexual transmission at mucosal surfaces. Partial efficacy (31.2%) of the Thai RV144 HIV-1 vaccine trial has been correlated with Antibody-dependent Cellular Cytotoxicity (ADCC) mediated by non-neutralizing antibodies targeting the V1V2 region of the HIV-1 envelope. This has led to speculation that ADCC and other antibody-dependent cellular effector functions might provide an important defense against mucosal acquisition of HIV-1 infection. However, the ability of antibody-dependent cellular effector mechanisms to impact on early mucosal transmission events will depend on a variety of parameters including effector cell type, frequency, the class of Fc-Receptor (FcR) expressed, the number of FcR per cell and the glycoslyation pattern of the induced antibodies. In this study, we characterize and compare the frequency and phenotype of IgG (CD16 [FcγRIII], CD32 [FcγRII] and CD64 [FcγRI]) and IgA (CD89 [FcαR]) receptor expression on effector cells within male and female genital mucosal tissue, colorectal tissue and red blood cell-lysed whole blood. The frequency of FcR expression on CD14+ monocytic cells, myeloid dendritic cells and natural killer cells were similar across the three mucosal tissue compartments, but significantly lower when compared to the FcR expression profile of effector cells isolated from whole blood, with many cells negative for all FcRs. Of the three tissues tested, penile tissue had the highest percentage of FcR positive effector cells. Immunofluorescent staining was used to determine the location of CD14+, CD11c+ and CD56+ cells within the three mucosal tissues. We show that the majority of effector cells across the different mucosal locations reside within the subepithelial lamina propria. The potential implication of the observed FcR expression patterns on the effectiveness of FcR-dependent cellular effector functions to impact on the ini
Kopycinski J, Hayes P, Ashraf A, et al., 2014, Broad HIV Epitope Specificity and Viral Inhibition Induced by Multigenic HIV-1 Adenovirus Subtype 35 Vector Vaccine in Healthy Uninfected Adults, PLOS ONE, Vol: 9, ISSN: 1932-6203
Hayes P, Gilmour J, von Lieven A, et al., 2013, Safety and immunogenicity of DNA prime and modified vaccinia ankara virus-HIV subtype C vaccine boost in healthy adults., Clin Vaccine Immunol, Vol: 20, Pages: 397-408
A randomized, double-blind, placebo-controlled phase I trial was conducted in 32 HIV-uninfected healthy volunteers to assess the safety and immunogenicity of 3 doses of DNA vaccine (Advax) plus 1 dose of recombinant modified vaccinia virus Ankara (MVA) (TBC-M4) or 3 doses of TBC-M4 alone (groups A and B, respectively). Both vaccine regimens were found to be safe and well tolerated. Gamma interferon (IFN-γ) enzyme-linked immunosorbent spot (ELISPOT) assay responses were detected in 1/10 (10%) individuals in group A after three Advax primes and in 9/9 individuals (100%) after the MVA boost. In group B, IFN-γ ELISPOT responses were detected in 6/12 (50%) and 7/11 (64%) individuals after the second and third MVA vaccinations, respectively. Responses to all vaccine components, but predominantly to Env, were seen. The breadth and magnitude of the T cell response and viral inhibition were greater in group A than in group B, indicating that the quality of the T-cell response was enhanced by the DNA prime. Intracellular cytokine staining indicated that the T-cell responses were polyfunctional but were skewed toward Env with a CD4(+) phenotype. At 2 weeks after the last vaccination, HIV-specific antibody responses were detected in all (100%) group B and 1/11 (9.1%) group A vaccinees. Vaccinia virus-specific responses were detected in all (100%) group B and 2/11 (18.2%) group A vaccinees. In conclusion, HIV-specific T-cell responses were seen in the majority of volunteers in groups A and B but with a trend toward greater quality of the T-cell response in group A. Antibody responses were better in group B than in group A.
Kopycinski J, Cheeseman H, Ashraf A, et al., 2012, A DNA-based candidate HIV vaccine delivered via in vivo electroporation induces CD4 responses toward the α4β7-binding V2 loop of HIV gp120 in healthy volunteers., Clin Vaccine Immunol, Vol: 19, Pages: 1557-1559
Administration of a clade C/B' candidate HIV-1 DNA vaccine, ADVAX, by in vivo electroporation (EP) was safe and more immunogenic than intramuscular administration without EP. The breadth and specificity of T-cell responses to full-length Env were mapped. Responses to multiple Env regions were induced, with most focusing on V3/C4 and V2 regions, including the α4β7 integrin-binding domain. The breadth of responses induced by this DNA vaccine regimen was comparable to that of viral-vectored vaccine regimens.
Keefer MC, Gilmour J, Hayes P, et al., 2012, A phase I double blind, placebo-controlled, randomized study of a multigenic HIV-1 adenovirus subtype 35 vector vaccine in healthy uninfected adults., PLoS One, Vol: 7
BACKGROUND: We conducted a phase I, randomized, double-blind, placebo-controlled trial to assess the safety and immunogenicity of escalating doses of two recombinant replication defective adenovirus serotype 35 (Ad35) vectors containing gag, reverse transcriptase, integrase and nef (Ad35-GRIN) and env (Ad35-ENV), both derived from HIV-1 subtype A isolates. The trial enrolled 56 healthy HIV-uninfected adults. METHODS: Ad35-GRIN/ENV (Ad35-GRIN and Ad35-ENV mixed in the same vial in equal proportions) or Ad35-GRIN was administered intramuscularly at 0 and 6 months. Participants were randomized to receive either vaccine or placebo (10/4 per group, respectively) within one of four dosage groups: Ad35-GRIN/ENV 2×10(9) (A), 2×10(10) (B), 2×10(11) (C), or Ad35-GRIN 1×10(10) (D) viral particles. RESULTS: No vaccine-related serious adverse event was reported. Reactogenicity events reported were dose-dependent, mostly mild or moderate, some severe in Group C volunteers, all transient and resolving spontaneously. IFN-γ ELISPOT responses to any vaccine antigen were detected in 50, 56, 70 and 90% after the first vaccination, and in 75, 100, 88 and 86% of Groups A-D vaccine recipients after the second vaccination, respectively. The median spot forming cells (SFC) per 10(6) PBMC to any antigen was 78-139 across Groups A-C and 158-174 in Group D, after each of the vaccinations with a maximum of 2991 SFC. Four to five HIV proteins were commonly recognized across all the groups and over multiple timepoints. CD4+ and CD8+ T-cell responses were polyfunctional. Env antibodies were detected in all Group A-C vaccinees and Gag antibodies in most vaccinees after the second immunization. Ad35 neutralizing titers remained low after the second vaccination. CONCLUSION/SIGNIFICANCE: Ad35-GRIN/ENV reactogenicity was dose-related. HIV-specific cellular and humoral responses were seen in the majority of volunteers immunized with Ad35-GRIN/ENV or Ad35-GRIN and increased
Kaltsidis H, Cheeseman H, Kopycinski J, et al., 2011, Measuring human T cell responses in blood and gut samples using qualified methods suitable for evaluation of HIV vaccine candidates in clinical trials., J Immunol Methods, Vol: 370, Pages: 43-54
The next generation of candidate HIV vaccines include replicating vectors selected for tropism to mucosal sites, where an efficacious T cell response will be required to limit T cell replication and HIV associated CD4 T cell loss. To fully assess immunogenicity of such candidates, there is a need to develop robust quality controlled analysis of gut derived HIV specific CD8+ T-cell responses. Despite obvious challenges in obtaining sufficient amounts of tissue, the highly compartmentalised nature of the mucosal immune responses, requires the assessment of CD8 T cells isolated directly from local tissue before any conclusions regarding the induction of mucosal responses are made. Here we describe the optimisation and subsequent qualification of a qualitative and quantitative polychromatic flow cytometry assay to assess antigen specific CD8+ T cells isolated from the gut, using samples from HIV positive and negative volunteers. Internal quality controls monitored over time, combined with the use of quality gating and standard operating procedures were used to demonstrate the generation of robust and reliable data.
Vasan S, Hurley A, Schlesinger SJ, et al., 2011, In vivo electroporation enhances the immunogenicity of an HIV-1 DNA vaccine candidate in healthy volunteers., PLoS One, Vol: 6
BACKGROUND: DNA-based vaccines have been safe but weakly immunogenic in humans to date. METHODS AND FINDINGS: We sought to determine the safety, tolerability, and immunogenicity of ADVAX, a multigenic HIV-1 DNA vaccine candidate, injected intramuscularly by in vivo electroporation (EP) in a Phase-1, double-blind, randomized placebo-controlled trial in healthy volunteers. Eight volunteers each received 0.2 mg, 1 mg, or 4 mg ADVAX or saline placebo via EP, or 4 mg ADVAX via standard intramuscular injection at weeks 0 and 8. A third vaccination was administered to eleven volunteers at week 36. EP was safe, well-tolerated and considered acceptable for a prophylactic vaccine. EP delivery of ADVAX increased the magnitude of HIV-1-specific cell mediated immunity by up to 70-fold over IM injection, as measured by gamma interferon ELISpot. The number of antigens to which the response was detected improved with EP and increasing dosage. Intracellular cytokine staining analysis of ELISpot responders revealed both CD4+ and CD8+ T cell responses, with co-secretion of multiple cytokines. CONCLUSIONS: This is the first demonstration in healthy volunteers that EP is safe, tolerable, and effective in improving the magnitude, breadth and durability of cellular immune responses to a DNA vaccine candidate. TRIAL REGISTRATION: ClinicalTrials.gov NCT00545987.
Vasan S, Hurley A, Schlesinger SJ, et al., 2011, In vivo electroporation enhances the immunogenicity of an HIV-1 DNA vaccine candidate in healthy volunteers, PLoS One, Vol: 6, ISSN: 1932-6203
Spentzou A, Bergin P, Gill D, et al., 2010, Viral inhibition assay: a CD8 T cell neutralization assay for use in clinical trials of HIV-1 vaccine candidates., J Infect Dis, Vol: 201, Pages: 720-729
We have characterized an assay measuring CD8 T cell-mediated inhibition of human immunodeficiency virus (HIV) type 1 replication, demonstrating specificity and reproducibility and employing a panel of primary HIV-1 isolates. The assay uses relatively simple autologous cell culture and enzyme-linked immunosorbent assay, avoids generation of T cell clones, and can be performed with <2 million peripheral blood mononuclear cells. Efficient CD8 T cell-mediated cross-clade inhibition of HIV-1 replication in vitro was demonstrated in antiretroviral therapy-naive HIV-1-infected subjects with controlled viral replication in vivo but not in viremic subjects. An HIV-1 vaccine candidate, consisting of DNA and recombinant adenovirus 5 vectors tested in a phase I clinical trial, induced CD8 T cells that efficiently inhibited HIV-1 in a HLA-I-dependent manner. Assessment of direct antiviral T cell function by this assay provides additional information to guide vaccine design and the prioritizing of candidates for further clinical trials.
Vasan S, Hurley A, Schlesinger SJ, et al., 2009, OA05-01. In vivo electroporation enhances the immunogenicity of ADVAX, a DNA-based HIV-1 vaccine candidate, in healthy volunteers, Retrovirology, Vol: 6
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