Antigen Discovery (United States)

The World Health Organisation has identified Acinetobacter baumannii as a critical priority antimicrobial resistant (AMR) pathogen for which new therapeutics are needed. Despite this, currently there are no antibody or vaccine candidates in advanced clinical development for A. baumannii. To help address this, we designed a protein microarray approach to identify multiple A. baumannii protein antigens for further investigation as potential targets for vaccination or an antibody therapy. An 868-protein microarray was constructed containing mainly highly conserved A. baumannii proteins, and was enriched for those predicted to be surface localised and for which the corresponding gene is highly expressed during culture in ex vivo human serum. Probing the protein microarray with sera obtained from mice after non-lethal infection with multiple different A. baumannii strains identified IgG responses to 66 proteins. Four proteins (three previously poorly described outer membrane proteins and BamA, a known protective vaccine antigen selected as a positive control) were selected for further investigation. Polyclonal rabbit IgG to all four protein antigens recognised multiple clinical AMR A. baumannii strains, and for selected strains promoted opsonisation with IgG and complement, improved neutrophil phagocytosis, and increased membrane attack complex formation. Passive immunisation with polyclonal IgG to each antigen partially protected mice against A. baumannii sepsis, and a combination of polyclonal to two antigens completely protected against A. baumannii murine sepsis. Repeating passive immunisation experiments in mice depleted of complement, neutrophils or tissue macrophages demonstrated protection against systemic infection was dependent on complement and neutrophils but not macrophages. Overall, the data demonstrate that our protein microarray is a novel approach that can rapidly identify multiple new protein antigens as potential antibody targets for preventing or treating AMR bacterial infections.

Shigella is a leading cause of diarrheal morbidity and mortality in young children from low- and middle-income countries. Here, we aimed to verify the ability of the generalized modules for membrane antigens (GMMA)-based Shigella sonnei candidate vaccine 1790GAHB to elicit an anti-protein antibody response. Serum samples from previous clinical trials in adults (a dose-escalation study and its extension in France, a vaccine efficacy study after human challenge in the United States, and a study in Kenya) were investigated using pan-proteome microarrays consisting of 3,150 full-length or fragmented Shigella proteins. Pre-/post-vaccination comparisons identified subsets of proteins that were highly immunoreactive and largely overlapped across all trials; the T3SS lipochaperone family protein (expressed on GMMA) was the most reactive in all studies. Responses to several microarray antigens correlated well with S. sonnei LPS serum IgG antibody levels. Overall, we confirmed the ability of GMMA to elicit an anti-protein IgG/IgA response; however, no association with protection against shigellosis was identified. In the challenge study, IgG response to seven antigens (IpaC, IpaB, IpaA, IpaD, IpaH, IpgC, and MxiD; not expressed on GMMA) was associated with a decreased risk of shigellosis. These antigens were observed to also have high IgG responses at baseline in individuals naturally exposed to Shigella and could constitute targets for future vaccine development.IMPORTANCEShigella remains a major cause of diarrheal disease, especially in children aged under 5 years from low-to-middle-income countries. No vaccine against shigellosis is yet widely available despite the high public health need. An ideal vaccine would provide protection against the most prevalent species, Shigella flexneri and Shigella sonnei; therefore, it could be relevant to identify common antigens. We developed a microarray containing 3,150 full-length or fragmented proteins selected across Shigella species. Sera collected in four clinical trials conducted in three countries of varying endemicity to evaluate a S. sonnei GMMA-based candidate vaccine were tested against these proteins. We identified several Shigella proteins (IpaC, IpaB, IpaA, IpaD, IpaH, IpgC, MxiD) that induced robust antibody response following experimental challenge or natural infection. These proteins correlated with a reduced risk of shigellosis after the S. sonnei challenge. We found no apparent role for anti-GMMA proteins' IgG or IgA response in protection against shigellosis.

Whooping cough (pertussis) is a highly contagious respiratory disease caused by the Gram-negative bacterium, Bordetella pertussis. Globally, tens of millions of whole-cell pertussis (wP) vaccines are administered annually. Whole-cell pertussis vaccines are logistically complex to manufacture and get to market because of the need for each batch of vaccine to be evaluated in a highly laborious and challenging potency test known as the Kendrick assay, which involves mouse intracerebral challenges with B. pertussis. In this report, we describe efforts to develop a serology-based substitute for the Kendrick assay that relies on profiling antibody responses to wP vaccines.

Abstract RationalePneumococcal pneumonia remains a global health problem. Pneumococcal colonisation increases local and systemic protective immunity, suggesting nasal administration of live attenuatedS. pneumoniaestrains could help prevent infections.ObjectivesWe used a controlled human infection model to investigate whether nasopharyngeal colonisation with attenuatedS. pneumoniaestrains protected against re-colonisation with wild-type (WT)S. pneumoniae(Spn).MethodsHealthy adults aged 18-50 years were randomised (1:1:1:1) for nasal administration twice (two weeks interval) with saline, WT Spn6B (BHN418) or one of two genetically modified Spn6B strains - SpnA1 (Δfhs/piaA) or SpnA3 (ΔproABC/piaA) (Stage I). After 6 months, participants were challenged with SpnWT to assess protection against re-colonisation (Stage II).Measurements and Main Results125 participants completed both study stages as per intention to treat. No Serious Adverse Events were reported. In Stage I, colonisation rates were similar amongst groups: SpnWT 58.1% (18/31), SpnA1 60% (18/30) and SpnA3 59.4% (19/32). Anti-Spn nasal IgG levels post-colonisation were similar in all groups whilst serum IgG responses were higher in the SpnWT and SpnA1 groups than the SpnA3 group. In colonised individuals, increases in IgG responses were identified against 197 Spn protein antigens and serotype 6 capsular polysaccharide using a pangenome array. Participants given SpnWT or SpnA1 but not SpnA3 in phase 1 were partially protected against re-colonisation with SpnWT (recolonisation rates of 29% versus 30% respectively).ConclusionNasal colonisation with genetically modified live attenuated Spn was safe and induced protection against recolonisation, suggesting nasal adminstration of live attenuated Spn could be an effective stategy for preventing pneumococcal infections.

BackgroundOne potential mechanism for protection from SARS-CoV-2 in children is through passive immunity via breast milk from a mother infected with the novel coronavirus. The primary objectives of this study were to establish the presence of SARS-CoV-2-specific IgA and IgG and to characterize the antigenic regions of SARS-CoV-2 proteins that were reactive with antibodies in breast milk.MethodsBetween March 2020 and September 2020, 21 women with confirmed SARS-CoV-2 infection were enrolled in Mommy’s Milk. Participants donated serial breast milk samples around their time of illness. Breast milk samples were used to probe a multi-coronavirus protein microarray containing full-length and variable-length overlapping fragments of SARS-CoV-2 proteins. Samples were also tested against S and N proteins by electrochemiluminescence assay.ResultsThe breast milk samples contained IgA reactive with a variety of SARS-CoV-2 antigens. The most IgA-reactive SARS-CoV-2 proteins were N (42.9% of women responded to ≥1 N fragment) and S proteins (23.9% responded to ≥1 fragment of S1 or S2). IgG responses were similar. A striking observation was the dissimilarity between mothers in antibody recognition, giving distinct antibody reactivity and kinetic profiles.ConclusionsIndividual COVID-19 cases had diverse and unique milk IgA profiles following the onset of symptoms.ImpactIn this observational longitudinal case series of 21 women with confirmed SARS-CoV-2 infection, IgA binding to SARS-CoV-2 proteins detected by orthologous proteome microarray and electrochemiluminescence assays was observed in >75% of women, but there was heterogeneity in which antigens and how many were reactive between women.Immunological profiles of protein regions recognized by each woman were distinct.Diverse repertoires of mucosal breast milk antibody to SARS-CoV-2 reflect heterogeneous passive transfer of maternal antibody to exposed breastfeeding infants.

Exacerbations of symptoms represent an unmet need for people with asthma. Bacterial dysbiosis and opportunistic bacterial infections have been observed in, and may contribute to, more severe asthma. However, the molecular mechanisms driving these exacerbations remain unclear. We show here that bacterial lipopolysaccharide (LPS) induces oncostatin M (OSM) and that airway biopsies from patients with severe asthma present with an OSM-driven transcriptional profile. This profile correlates with activation of inflammatory and mucus-producing pathways. Using primary human lung tissue or human epithelial and mesenchymal cells, we demonstrate that OSM is necessary and sufficient to drive pathophysiological features observed in severe asthma after exposure to LPS or Klebsiella pneumoniae. These findings were further supported through blockade of OSM with an OSM-specific antibody. Single-cell RNA sequencing from human lung biopsies identified macrophages as a source of OSM. Additional studies using Osm-deficient murine macrophages demonstrated that macrophage-derived OSM translates LPS signals into asthma-associated pathologies. Together, these data provide rationale for inhibiting OSM to prevent bacterial-associated progression and exacerbation of severe asthma.

ABSTRACTThe rapid worldwide spread of SARS-CoV-2 has accelerated research and development for controlling the COVID-19 pandemic. A multi-coronavirus protein microarray was created containing full-length proteins, overlapping protein fragments of various lengths, and peptide libraries from SARS-CoV-2 and four other human coronaviruses. Sera from confirmed COVID-19 patients as well as unexposed individuals were applied to multicoronavirus arrays to identify specific antibody reactivity. High-level IgG, IgM, and IgA reactivity to structural proteins S, M, and N of SARS-CoV-2, as well as accessory proteins such as ORF3a and ORF7a, were observed that were specific to COVID-19 patients. Antibody reactivity against overlapping 100-, 50-, and 30-amino acid fragments of SARS-CoV-2 proteins was used to identify antigenic regions. Numerous proteins of SARS-CoV, Middle East respiratory syndrome coronavirus (MERS-CoV), and the endemic human coronaviruses HCoV-NL63 and HCoV-OC43 were also more reactive with IgG, IgM, and IgA in COVID-19 patient sera than in unexposed control sera, providing further evidence of immunologic cross-reactivity between these viruses. Whereas unexposed individuals had minimal reactivity against SARS-CoV-2 proteins that poorly correlated with reactivity against HCoV-NL63 and HCoV-OC43 S2 and N proteins, COVID-19 patient sera had higher correlation between SARS-CoV-2 and HCoV responses, suggesting that de novo antibodies against SARS-CoV-2 cross-react with HCoV epitopes. Array responses were compared with validated spike protein-specific IgG enzyme-linked immunosorbent assays (ELISAs), showing agreement between orthologous methods. SARS-CoV-2 microneutralization titers were low in the COVID-19 patient sera but correlated with array responses against S and N proteins. The multi-coronavirus protein microarray is a useful tool for mapping antibody reactivity in COVID-19 patients.IMPORTANCE With novel mutant SARS-CoV-2 variants of concern on the rise, knowledge of immune specificities against SARS-CoV-2 proteins is increasingly important for understanding the impact of structural changes in antibody-reactive protein epitopes on naturally acquired and vaccine-induced immunity, as well as broader topics of cross-reactivity and viral evolution. A multi-coronavirus protein microarray used to map the binding of COVID-19 patient antibodies to SARS-CoV-2 proteins and protein fragments as well as to the proteins of four other coronaviruses that infect humans has shown specific regions of SARS-CoV-2 proteins that are highly reactive with patient antibodies and revealed cross-reactivity of these antibodies with other human coronaviruses. These data and the multi-coronavirus protein microarray tool will help guide further studies of the antibody response to COVID-19 and to vaccination against this worldwide pandemic.

To identify immunodominant antigens that elicit a humoral immune response following a primary and a secondary genital infection, rhesus monkeys were inoculated cervically with Chlamydia trachomatis serovar D. Serum samples were collected and probed with a protein microarray expressing 864/894 (96.4%) of the open reading frames of the C. trachomatis serovar D genome. The antibody response to the primary infection was analyzed in 72 serum samples from 12 inoculated monkeys. The following criteria were utilized to identify immunodominant antigens: proteins found to be recognized by at least 75% (9/12) of the infected monkeys with at least 15% elevations in signal intensity from week 0 to week 8 post infection. All infected monkeys developed Chlamydia specific serum antibodies. Eight proteins satisfied the selection criteria for immunodominant antigens: CT242 (OmpH-like protein), CT541 (mip), CT681 (ompA), CT381 (artJ), CT443 (omcB), CT119 (incA), CT486 (fliY), and CT110 (groEL). Of these, three antigens, CT119, CT486 and CT381, were not previously identified as immunodominant antigens using non-human primate sera. Following the secondary infection, the antibody responses to the eight immunodominant antigens were analyzed and found to be quite different in intensity and duration to the primary infection. In conclusion, these eight immunodominant antigens can now be tested for their ability to identify individuals with a primary C. trachomatis genital infection and to design vaccine strategies to protect against a primary infection with this pathogen.

Breastfeeding provides defense against infectious disease during early life. The mechanisms underlying this protection are complex but likely include the vast array of immune cells and components, such as immunoglobulins, in milk. Simply characterizing the concentrations of these bioactives, however, provides only limited information regarding their potential relationships with disease risk in the recipient infant. Rather, understanding pathogen and antigen specificity profiles of milk-borne immunoglobulins might lead to a more complete understanding of how maternal immunity impacts infant health and wellbeing. Milk produced by women living in 11 geographically dispersed populations was applied to a protein microarray containing antigens from 16 pathogens, including diarrheagenic E. coli, Shigella spp., Salmonella enterica serovar Typhi, Staphylococcus aureus, Streptococcus pneumoniae, Mycobacterium tuberculosis and other pathogens of global health concern, and specific IgA and IgG binding was measured. Our analysis identified novel disease-specific antigen responses and suggests that some IgA and IgG responses vary substantially within and among populations. Patterns of antibody reactivity analyzed by principal component analysis and differential reactivity analysis were associated with either lower-to-middle-income countries (LMICs) or high-income countries (HICs). Antibody levels were generally higher in LMICs than HICs, particularly for Shigella and diarrheagenic E. coli antigens, although sets of S. aureus, S. pneumoniae, and some M. tuberculosis antigens were more reactive in HICs. Differential responses were typically specific to canonical immunodominant antigens, but a set of nondifferential but highly reactive antibodies were specific to antigens possibly universally recognized by antibodies in human milk. This approach provides a promising means to understand how breastfeeding and human milk protect (or do not protect) infants from environmentally relevant pathogens. Furthermore, this approach might lead to interventions to boost population-specific immunity in at-risk breastfeeding mothers and their infants.