Local and Systemic Immunity against Respiratory Syncytial Virus Induced by a Novel Intranasal Vaccine. A Randomized, Double-Blind, Placebo-controlled Clinical Trial

RATIONALE:Needle-free intranasal vaccines offer major potential advantages, especially against pathogens entering via mucosal surfaces. As yet, there is no effective vaccine against respiratory syncytial virus (RSV), a ubiquitous pathogen of global importance that preferentially infects respiratory epithelial cells; new strategies are urgently required. OBJECTIVES:Here, we report the safety and immunogenicity of a novel mucosal RSV F protein vaccine linked to an immunostimulatory bacterium-like particle (BLP). METHODS:In this phase I, randomised, double-blind placebo-controlled trial, 48 healthy volunteers aged 18-49 years were randomly assigned to receive placebo or SynGEM (low- or high-dose) intranasally by prime-boost administration. The primary outcome was safety and tolerability, with secondary objectives assessing virus-specific immunogenicity. MEASUREMENTS AND MAIN RESULTS:There were no significant differences in adverse events between placebo and vaccinated groups. SynGEM induced systemic plasmablast responses and significant, durable increases in RSV-specific serum antibody in healthy seropositive adults. Volunteers given low-dose SynGEM (140 µg F, 2mg BLP) required a boost at day 28 to achieve plateau responses with a maximum fold-change of 2.4, whereas high-dose recipients (350 µg F, 5mg BLP) achieved plateau responses with a fold-change of 1.5 after first vaccination that remained elevated up to 180 days post-vaccination irrespective of further boosting. Palivizumab-like antibodies were consistently induced, but F protein site Ø-specific antibodies were not detected and virus-specific nasal IgA responses were heterogeneous, with strongest responses in individuals with lower pre-existing antibody levels. CONCLUSIONS:SynGEM is thus the first non-replicating intranasal RSV subunit vaccine to induce persistent antibody responses in human volunteers. Clinical trial registration available at www.clinicaltrials.gov, ID NCT02958540

Inflammatory profiles across the spectrum of disease reveal a distinct role for GM-CSF in severe COVID-19

While it is now widely accepted that host inflammatory responses contribute to lung injury, the pathways that drive severity and distinguish coronavirus disease 2019 (COVID-19) from other viral lung diseases remain poorly characterized. We analyzed plasma samples from 471 hospitalized patients recruited through the prospective multicenter ISARIC4C study and 39 outpatients with mild disease, enabling extensive characterization of responses across a full spectrum of COVID-19 severity. Progressive elevation of levels of numerous inflammatory cytokines and chemokines (including IL-6, CXCL10, and GM-CSF) were associated with severity and accompanied by elevated markers of endothelial injury and thrombosis. Principal component and network analyses demonstrated central roles for IL-6 and GM-CSF in COVID-19 pathogenesis. Comparing these profiles to archived samples from patients with fatal influenza, IL-6 was equally elevated in both conditions whereas GM-CSF was prominent only in COVID-19. These findings further identify the key inflammatory, thrombotic, and vascular factors that characterize and distinguish severe and fatal COVID-19

Large-scale phenotyping of patients with long COVID post-hospitalization reveals mechanistic subtypes of disease

One in ten severe acute respiratory syndrome coronavirus 2 infections result in prolonged symptoms termed long coronavirus disease (COVID), yet disease phenotypes and mechanisms are poorly understood1. Here we profiled 368 plasma proteins in 657 participants ≥3 months following hospitalization. Of these, 426 had at least one long COVID symptom and 233 had fully recovered. Elevated markers of myeloid inflammation and complement activation were associated with long COVID. IL-1R2, MATN2 and COLEC12 were associated with cardiorespiratory symptoms, fatigue and anxiety/depression; MATN2, CSF3 and C1QA were elevated in gastrointestinal symptoms and C1QA was elevated in cognitive impairment. Additional markers of alterations in nerve tissue repair (SPON-1 and NFASC) were elevated in those with cognitive impairment and SCG3, suggestive of brain–gut axis disturbance, was elevated in gastrointestinal symptoms. Severe acute respiratory syndrome coronavirus 2-specific immunoglobulin G (IgG) was persistently elevated in some individuals with long COVID, but virus was not detected in sputum. Analysis of inflammatory markers in nasal fluids showed no association with symptoms. Our study aimed to understand inflammatory processes that underlie long COVID and was not designed for biomarker discovery. Our findings suggest that specific inflammatory pathways related to tissue damage are implicated in subtypes of long COVID, which might be targeted in future therapeutic trials

Analysis of the CD4+ T cell immune response to Anthrax Lethal Factor

The Gram-positive bacterium Bacillus anthracis is the causative agent of the potentially fatal illness, anthrax. A major determinant of B. anthracis pathogenicity is a binary toxin composed of Protective Antigen (PA) and one of two subunits; Lethal Factor (LF) or Edema Factor (EF). Translocation by PA into the host cell cytosol allows LF, which is a zinc metalloprotease, to inactivate the mitogen-activated protein kinase (MAPK) pathway. The expected consequences for the immune response include subversion of antigen presentation and T cell priming. In contrast to this, long term CD4+ T cell responses to LF were identified following natural human anthrax infection and vaccination, indicating that this toxin component is a principle B. anthracis antigen which may confer protective immunity. It has been observed that polymorphism in the HLA class II alleles at the DR and DQ loci affects susceptibilty to infectious disease outcome. In order to map the human response within a defined genetic background, transgenic mice expressing individual HLA heterodimers in the absence of endogenous MHC class II were utilised. HLA-DR1, HLA-DR15, HLA-DR4, HLA-DQ8 and HLA-DQ6 (the latter generated as part of this project) transgenic mice were compared in terms of response magnitude to LF and HLA expression levels. This was correlated with survival following live anthrax challenge. Immunodominant epitopes within LF were elucidated for all HLA-transgenic lines. Immunogenicity in the transgenic model was shown to be primarily restricted to epitopes from domains II and IV. Dominant epitopes, which were common to all HLA types, were identified in domain II. HLA-DR specific epitopes were also identified. T cell responses to cryptic epitopes, revealed following immunisation with the individual peptides, were compared to the immunodominant epitope hierarchy. A peptide epitope of LF was identified with a strong relative binding affinity for HLA-DR15, making it a candidate therapeutic for ‘MHC blockade’ strategies. This epitope was tested for therapeutic blockade in a humanised transgenic mouse model which develops spontaneous paralysis with central nervous system (CNS) pathology similar to human multiple sclerosis (MS). Disease is characterised by an autoimmune response to a DR15 restricted epitope of myelin basic protein (MBP). The LF peptide was found to prevent induction of antigen-specific T cell responses, presumably by HLA class II, reducing the inflammatory response to self-peptide both in vitro and in vivo

Impact of HLA Polymorphism on the Immune Response to Bacillus Anthracis Protective Antigen in Vaccination versus Natural Infection

The causative agent of anthrax, Bacillus anthracis, evades the host immune response and establishes infection through the production of binary exotoxins composed of Protective Antigen (PA) and one of two subunits, lethal factor (LF) or edema factor (EF). The majority of vaccination strategies have focused upon the antibody response to the PA subunit. We have used a panel of humanised HLA class II transgenic mouse strains to define HLA-DR-restricted and HLA-DQ-restricted CD4+ T cell responses to the immunodominant epitopes of PA. This was correlated with the binding affinities of epitopes to HLA class II molecules, as well as the responses of two human cohorts: individuals vaccinated with the Anthrax Vaccine Precipitated (AVP) vaccine (which contains PA and trace amounts of LF), and patients recovering from cutaneous anthrax infections. The infected and vaccinated cohorts expressing different HLA types were found to make CD4+ T cell responses to multiple and diverse epitopes of PA. The effects of HLA polymorphism were explored using transgenic mouse lines, which demonstrated differential susceptibility, indicating that HLA-DR1 and HLA-DQ8 alleles conferred protective immunity relative to HLA-DR15, HLA-DR4 and HLA-DQ6. The HLA transgenics enabled a reductionist approach, allowing us to better define CD4+ T cell epitopes. Appreciating the effects of HLA polymorphism on the variability of responses to natural infection and vaccination is vital in planning protective strategies against anthrax

Natural cutaneous anthrax infection, but not vaccination, induces a CD4⁺ T cell response involving diverse cytokines

BACKGROUND: Whilst there have been a number of insights into the subsets of CD4(+) T cells induced by pathogenic Bacillus anthracis infections in animal models, how these findings relate to responses generated in naturally infected and vaccinated humans has yet to be fully established. We describe the cytokine profile produced in response to T cell stimulation with a previously defined immunodominant antigen of anthrax, lethal factor (LF), domain IV, in cohorts of individuals with a history of cutaneous anthrax, compared with vaccinees receiving the U.K. licenced Anthrax Vaccine Precipitated (AVP) vaccine. FINDINGS: We found that immunity following natural cutaneous infection was significantly different from that seen after vaccination. AVP vaccination was found to result in a polarized IFNγ CD4+ T cell response, while the individuals exposed to B. anthracis by natural infection mounted a broader cytokine response encompassing IFNγ, IL-5, −9, −10, −13, −17, and −22. CONCLUSIONS: Vaccines seeking to incorporate the robust, long-lasting, CD4 T cell immune responses observed in naturally acquired cutaneous anthrax cases may need to elicit a similarly broad spectrum cellular immune response

CD4+ T Cells Targeting Dominant and Cryptic Epitopes from Bacillus anthracis Lethal Factor

Anthrax is an endemic infection in many countries, particularly in the developing world. The causative agent, Bacillus anthracis, mediates disease through the secretion of binary exotoxins. Until recently, research into adaptive immunity targeting this bacterial pathogen has largely focused on the humoral response to these toxins. There is, however, growing recognition that cellular immune responses involving IFNγ producing CD4+ T cells also contribute significantly to a protective memory response. An established concept in adaptive immunity to infection is that during infection of host cells, new microbial epitopes may be revealed, leading to immune recognition of so called ‘cryptic’ or ‘subdominant’ epitopes. We analyzed the response to both cryptic and immunodominant T cell epitopes derived from the toxin component lethal factor and presented by a range of HLA-DR alleles. Using IFNγ-ELISpot assays we characterized epitopes that elicited a response following immunization with synthetic peptide and the whole protein and tested their capacities to bind purified HLA-DR molecules in vitro. We found that DR1 transgenics demonstrated T cell responses to a greater number of domain III cryptic epitopes than other HLA-DR transgenics, and that this pattern was repeated with the immunodominant epitopes, as a greater proportion of these epitopes induced a T cell response when presented within the context of the whole protein. Immunodominant epitopes LF457-476 and LF467-487 were found to induce a T cell response to the peptide, as well as to the whole native LF protein in DR1 and DR15, but not in DR4 transgenics. The analysis of Domain I revealed the presence of several unique cryptic epitopes all of which showed a strong to moderate relative binding affinity to HLA-DR4 molecules. However, none of the cryptic epitopes from either domain III or I displayed notably high binding affinities across all HLA-DR alleles assayed. These responses were influenced by the specific HLA alleles presenting the peptide, and imply that construction of future epitope string vaccines which are immunogenic across a wide range of HLA alleles could benefit from a combination of both cryptic and immunodominant anthrax epitopes