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Coxiella burnetii is the causative agent of Q fever, for which there is yet to be an FDA-approved vaccine. This bacterial pathogen has both extra- and intracellular stages in its life cycle, and therefore both a cell-mediated (i.e., T lymphocyte) and humoral (i.e., antibody) immune response are necessary for effective eradication of this pathogen. However, most proposed vaccines elicit strong responses to only one mechanism of adaptive immunity, and some can either cause reactogenicity or lack sufficient immunogenicity. In this work, we aim to apply a nanoparticle-based platform toward producing both antibody and T cell immune responses against C. burnetii. We investigated three approaches for conjugation of the immunodominant outer membrane protein antigen (CBU1910) to the E2 nanoparticle to obtain a consistent antigen orientation: direct genetic fusion, high affinity tris-NTA-Ni conjugation to polyhistidine-tagged CBU1910, and the SpyTag/SpyCatcher (ST/SC) system. Overall, we found that the ST/SC approach yielded nanoparticles loaded with the highest number of antigens while maintaining stability, enabling formulations that could simultaneously co-deliver the protein antigen (CBU1910) and adjuvant (CpG1826) on one nanoparticle (CBU1910-CpG-E2). Using protein microarray analyses, we found that after immunization, antigen-bound nanoparticle formulations elicited significantly higher antigen-specific IgG responses than soluble CBU1910 alone and produced more balanced IgG1/IgG2c ratios. Although T cell recall assays from these protein antigen formulations did not show significant increases in antigen-specific IFN-γ production compared to soluble CBU1910 alone, nanoparticles conjugated with a CD4 peptide epitope from CBU1910 generated elevated T cell responses in mice to both the CBU1910 peptide epitope and whole CBU1910 protein. These investigations highlight the feasibility of conjugating antigens to nanoparticles for tuning and improving both humoral- and cell-mediated adaptive immunity against C. burnetii.

IntroductionIn the context of recurrent surges of SARS-CoV-2 infections, a detailed characterization of antibody persistence over a 6-month period following vaccine booster dose is necessary to crafting effective public health policies on repeat vaccination.MethodsTo characterize the SARS-CoV-2 antibody profile of a healthcare worker population over a 6-month period following mRNA vaccination and booster dose. 323 healthcare workers at an academic medical center in Orange County, California who had completed primary vaccination and booster dose against SARS-CoV-2 were recruited for the study. A total of 690 blood specimens over a 6-month period were collected via finger-stick blood and analyzed for the presence of antibodies against 9 SARS-CoV-2 antigens using a coronavirus antigen microarray.ResultsThe primary outcome of this study was the average SARS-CoV-2 antibody level as measured using a novel coronavirus antigen microarray. Additional outcomes measured include levels of antibodies specific to SARS-CoV-2 variants including Delta, Omicron BA.1, and BA.2. We also measured SARS-CoV-2 neutralization capacity for a subset of the population to confirm correlation with antibody levels. Although antibodies against SARS-CoV-2 wane throughout the 6-month period following a booster dose, antibody levels remain higher than pre-boost levels. However, a booster dose of vaccine based on the original Wuhan strain generates approximately 3-fold lower antibody reactivity against Omicron variants BA.1 and BA.2 as compared to the vaccine strain. Despite waning antibody levels, neutralization activity against the vaccine strain is maintained throughout the 6-month period.DiscussionIn the context of recurrent surges of SARS-CoV-2 infections, our data indicate that breakthrough infections are likely driven by novel variants with different antibody specificity and not by time since last dose of vaccination, indicating that development of vaccinations specific to these novel variants is necessary to prevent future surges of SARS-CoV-2 infections.

BackgroundWhile others have reported severe acute respiratory syndrome-related coronavirus 2(SARS-CoV-2) seroprevalence studies in health care workers (HCWs), we leverage the use of a highly sensitive coronavirus antigen microarray to identify a group of seropositive health care workers who were missed by daily symptom screening that was instituted prior to any epidemiologically significant local outbreak. Given that most health care facilities rely on daily symptom screening as the primary method to identify SARS-CoV-2 among health care workers, here, we aim to determine how demographic, occupational, and clinical variables influence SARS-CoV-2 seropositivity among health care workers.MethodsWe designed a cross-sectional survey of HCWs for SARS-CoV-2 seropositivity conducted from May 15th to June 30th 2020 at a 418-bed academic hospital in Orange County, California. From an eligible population of 5,349 HCWs, study participants were recruited in two ways: an open cohort, and a targeted cohort. The open cohort was open to anyone, whereas the targeted cohort that recruited HCWs previously screened for COVID-19 or work in high-risk units. A total of 1,557 HCWs completed the survey and provided specimens, including 1,044 in the open cohort and 513 in the targeted cohort. Demographic, occupational, and clinical variables were surveyed electronically. SARS-CoV-2 seropositivity was assessed using a coronavirus antigen microarray (CoVAM), which measures antibodies against eleven viral antigens to identify prior infection with 98% specificity and 93% sensitivity.ResultsAmong tested HCWs (n = 1,557), SARS-CoV-2 seropositivity was 10.8%, and risk factors included male gender (OR 1.48, 95% CI 1.05-2.06), exposure to COVID-19 outside of work (2.29, 1.14-4.29), working in food or environmental services (4.85, 1.51-14.85), and working in COVID-19 units (ICU: 2.28, 1.29-3.96; ward: 1.59, 1.01-2.48). Amongst 1,103 HCWs not previously screened, seropositivity was 8.0%, and additional risk factors included younger age (1.57, 1.00-2.45) and working in administration (2.69, 1.10-7.10).ConclusionSARS-CoV-2 seropositivity is significantly higher than reported case counts even among HCWs who are meticulously screened. Seropositive HCWs missed by screening were more likely to be younger, work outside direct patient care, or have exposure outside of work.

BackgroundCOVID-19 convalescent plasma (CCP), from donors recovered from severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection, is one of the limited therapeutic options currently available for the treatment of critically ill patients with COVID-19. There is growing evidence that CCP may reduce viral loads and disease severity; and reduce mortality. However, concerns about the risk of transfusion-transmitted infections (TTI) and other complications associated with transfusion of plasma, remain. Amotosalen/UVA pathogen reduction treatment (A/UVA-PRT) of plasma offers a mitigation of TTI risk, and when combined with pooling has the potential to increase the diversity of the polyclonal SARS-CoV-2 neutralizing antibodies.Study design and methodsThis study assessed the impact of A/UVA-PRT on SARS-CoV-2 antibodies in 42 CCP using multiple complimentary assays including antigen binding, neutralizing, and epitope microarrays. Other mediators of CCP efficacy were also assessed.ResultsA/UVA-PRT did not negatively impact antibodies to SARS-CoV-2 and other viral epitopes, had no impact on neutralizing activity or other potential mediators of CCP efficacy. Finally, immune cross-reactivity with other coronavirus antigens was observed raising the potential for neutralizing activity against other emergent coronaviruses.ConclusionThe findings of this study support the selection of effective CCP combined with the use of A/UVA-PRT in the production of CCP for patients with COVID-19.

Recent studies provide conflicting evidence on the persistence of SARS-CoV-2 immunity induced by mRNA vaccines. Here, we aim to quantify the persistence of humoral immunity following vaccination using a coronavirus antigen microarray that includes 10 SARS-CoV-2 antigens. In a prospective longitudinal cohort of 240 healthcare workers, composite SARS-CoV-2 IgG antibody levels did not wane significantly over a 6-month study period. In the subset of the study population previously exposed to SARS-CoV-2 based on seropositivity for nucleocapsid antibodies, higher composite anti-spike IgG levels were measured before the vaccine but no significant difference from unexposed individuals was observed at 6 months. Age, vaccine type, or worker role did not significantly impact composite IgG levels, although non-significant trends towards lower antibody levels in older participants and higher antibody levels with Moderna vaccine were observed at 6 months. A small subset of our cohort were classified as having waning antibody titers at 6 months, and these individuals were less likely to work in patient care roles and more likely to have prior exposure to SARS-CoV-2.

Early in the SARS-CoV-2 pandemic, there was a high level of optimism based on observational studies and small controlled trials that treating hospitalized patients with convalescent plasma from COVID-19 survivors (CCP) would be an important immunotherapy. However, as more data from controlled trials became available, the results became disappointing, with at best moderate evidence of efficacy when CCP with high titers of neutralizing antibodies was used early in infection. To better understand the potential therapeutic efficacy of CCP, and to further validate SARS-CoV-2 infection of macaques as a reliable animal model for testing such strategies, we inoculated 12 adult rhesus macaques with SARS-CoV-2 by intratracheal and intranasal routes. One day later, 8 animals were infused with pooled human CCP with a high titer of neutralizing antibodies (RVPN NT50 value of 3,003), while 4 control animals received normal human plasma. Animals were monitored for 7 days. Animals treated with CCP had detectable but low levels of antiviral antibodies after infusion. In comparison to the control animals, CCP-treated animals had similar levels of viral RNA in upper and lower respiratory tract secretions, similar detection of viral RNA in lung tissues by in situ hybridization, but lower amounts of infectious virus in the lungs. CCP-treated animals had a moderate, but statistically significant reduction in interstitial pneumonia, as measured by comprehensive lung histology. Thus overall, therapeutic benefits of CCP were marginal and inferior to results obtained earlier with monoclonal antibodies in this animal model. By highlighting strengths and weaknesses, data of this study can help to further optimize nonhuman primate models to provide proof-of-concept of intervention strategies, and guide the future use of convalescent plasma against SARS-CoV-2 and potentially other newly emerging respiratory viruses.

The effects of adjuvants for increasing the immunogenicity of influenza vaccines are well known. However, the effect of adjuvants on increasing the breadth of cross-reactivity is less well understood. In this study we have performed a systematic screen of different toll-like receptor (TLR) agonists, with and without a squalene-in-water emulsion on the immunogenicity of a recombinant trimerized hemagglutinin (HA) vaccine in mice after single-dose administration. Antibody (Ab) cross-reactivity for other variants within and outside the immunizing subtype (homosubtypic and heterosubtypic cross-reactivity, respectively) was assessed using a protein microarray approach. Most adjuvants induced broad IgG profiles, although the response to a combination of CpG, MPLA and AddaVax (termed 'IVAX-1') appeared more quickly and reached a greater magnitude than the other formulations tested. Antigen-specific plasma cell labeling experiments show the components of IVAX-1 are synergistic. This adjuvant preferentially stimulates CD4 T cells to produce Th1>Th2 type (IgG2c>IgG1) antibodies and cytokine responses. Moreover, IVAX-1 induces identical homo- and heterosubtypic IgG and IgA cross-reactivity profiles when administered intranasally. Consistent with these observations, a single-cell transcriptomics analysis demonstrated significant increases in expression of IgG1, IgG2b and IgG2c genes of B cells in H5/IVAX-1 immunized mice relative to naïve mice, as well as significant increases in expression of the IFNγ gene of both CD4 and CD8 T cells. These data support the use of adjuvants for enhancing the breath and durability of antibody responses of influenza virus vaccines.

Effective methods for predicting COVID-19 disease trajectories are urgently needed. Here, enzyme-linked immunosorbent assay (ELISA) and coronavirus antigen microarray (COVAM) analysis mapped antibody epitopes in the plasma of COVID-19 patients (n = 86) experiencing a wide range of disease states. The experiments identified antibodies to a 21-residue epitope from nucleocapsid (termed Ep9) associated with severe disease, including admission to the intensive care unit (ICU), requirement for ventilators, or death. Importantly, anti-Ep9 antibodies can be detected within 6 days post-symptom onset and sometimes within 1 day. Furthermore, anti-Ep9 antibodies correlate with various comorbidities and hallmarks of immune hyperactivity. We introduce a simple-to-calculate, disease risk factor score to quantitate each patient's comorbidities and age. For patients with anti-Ep9 antibodies, scores above 3.0 predict more severe disease outcomes with a 13.42 likelihood ratio (96.7% specificity). The results lay the groundwork for a new type of COVID-19 prognostic to allow early identification and triage of high-risk patients. Such information could guide more effective therapeutic intervention.IMPORTANCE The COVID-19 pandemic has resulted in over two million deaths worldwide. Despite efforts to fight the virus, the disease continues to overwhelm hospitals with severely ill patients. Diagnosis of COVID-19 is readily accomplished through a multitude of reliable testing platforms; however, prognostic prediction remains elusive. To this end, we identified a short epitope from the SARS-CoV-2 nucleocapsid protein and also a disease risk factor score based upon comorbidities and age. The presence of antibodies specifically binding to this epitope plus a score cutoff can predict severe COVID-19 outcomes with 96.7% specificity.

Despite the enormous morbidity attributed to schistosomiasis, there is still no vaccine to combat the disease for the hundreds of millions of infected people. The anthelmintic drug, praziquantel, is the mainstay treatment option, although its molecular mechanism of action remains poorly defined. Praziquantel treatment damages the outermost surface of the parasite, the tegument, liberating surface antigens from dying worms that invoke a robust immune response which in some subjects results in immunologic resistance to reinfection. Herein we term this phenomenon Drug-Induced Vaccination (DIV). To identify the antigenic targets of DIV antibodies in urogenital schistosomiasis, we constructed a recombinant proteome array consisting of approximately 1,000 proteins informed by various secretome datasets including validated proteomes and bioinformatic predictions. Arrays were screened with sera from human subjects treated with praziquantel and shown 18 months later to be either reinfected (chronically infected subjects, CI) or resistant to reinfection (DIV). IgG responses to numerous antigens were significantly elevated in DIV compared to CI subjects, and indeed IgG responses to some antigens were completely undetectable in CI subjects but robustly recognized by DIV subjects. One antigen in particular, a cystatin cysteine protease inhibitor stood out as a unique target of DIV IgG, so recombinant cystatin was produced, and its vaccine efficacy assessed in a heterologous Schistosoma mansoni mouse challenge model. While there was no significant impact of vaccination with adjuvanted cystatin on adult worm numbers, highly significant reductions in liver egg burdens (45-55%, P

Combining variant antigens into a multivalent vaccine is a traditional approach used to provide broad coverage against antigenically variable pathogens, such as polio, human papilloma and influenza viruses. However, strategies for increasing the breadth of antibody coverage beyond the vaccine are not well understood, but may provide more anticipatory protection. Influenza virus hemagglutinin (HA) is a prototypic variant antigen. Vaccines that induce HA-specific neutralizing antibodies lose efficacy as amino acid substitutions accumulate in neutralizing epitopes during influenza virus evolution. Here we studied the effect of a potent combination adjuvant (CpG/MPLA/squalene-in-water emulsion) on the breadth and maturation of the antibody response to a representative variant of HA subtypes H1, H5 and H7. Using HA protein microarrays and antigen-specific B cell labelling, we show when administered individually, each HA elicits a cross-reactive antibody profile for multiple variants within the same subtype and other closely-related subtypes (homosubtypic and heterosubtypic cross-reactivity, respectively). Despite a capacity for each subtype to induce heterosubtypic cross-reactivity, broader coverage was elicited by simply combining the subtypes into a multivalent vaccine. Importantly, multiplexing did not compromise antibody avidity or affinity maturation to the individual HA constituents. The use of adjuvants to increase the breadth of antibody coverage beyond the vaccine antigens may help future-proof vaccines against newly-emerging variants.

A coronavirus antigen microarray (COVAM) was constructed containing 11 SARS-CoV-2, 5 SARS-1, 5 MERS, and 12 seasonal coronavirus recombinant proteins. The array is designed to measure immunoglobulin isotype and subtype levels in serum or plasma samples against each of the individual antigens printed on the array. We probed the COVAM with COVID-19 convalescent plasma (CCP) collected from 99 donors who recovered from a PCR+ confirmed SARS-CoV-2 infection. The results were analyzed using two computational approaches, a generalized linear model (glm) and random forest (RF) prediction model, to classify individual specimens as either Reactive or non-reactive against the SARS-CoV-2 antigens. A training set of 88 pre-COVID-19 specimens (PreCoV) collected in August 2019 and102 positive specimens from SARS-CoV-2 PCR+ confirmed COVID-19 cases was used for these analyses. Results compared with an FDA emergency use authorized (EUA) SARS-CoV2 S1-based total Ig chemiluminescence immunoassay (Ortho Clinical Diagnostics VITROS Anti-SARS-CoV-2 Total, CoV2T) and with a SARS-CoV-2 S1-S2 spike-based pseudovirus micro neutralization assay (SARS-CoV-2 reporter viral particle neutralization titration (RVPNT) showed high concordance between the three assays. Three CCP specimens that were negative by the VITROS CoV2T immunoassay were also negative by both COVAM and the RVPNT assay. Concordance between VITROS CoV2T and COVAM was 96%, VITROS CoV2T and RVPNT 93%, and RVPNT and COVAM 91%. The discordances were all weakly reactive samples near the cutoff threshold of the VITROS CoV2T immunoassay. The multiplex COVAM allows CCP to be grouped according to antibody reactivity patterns against 11 SARS-CoV-2 antigens. Unsupervised K-means analysis, via the gap statistics, as well as hierarchical clustering analysis revealed three main clusters with distinct reactivity intensities and patterns. These patterns were not recapitulated by adjusting the VITROS CoV2T or RVPNT assay thresholds. Plasma classified by COVAM reactivity patterns offers potential to improve CCP therapeutic efficacy CoV2T alone. The use of a SARS-CoV-2 antigen array can qualify CCP for administration as a treatment for acute COVID-19, and interrogate vaccine immunogenicity and performance in preclinical, clinical studies, and routine vaccination to identify antibody responses predictive of protection from infection and disease.

Antibodies against Aß amyloid are indispensable research tools and potential therapeutics for Alzheimer's disease. They display several unusual properties, such as specificity for aggregated forms of the peptide, the ability to distinguish polymorphic aggregate structures, and the ability to recognize generic aggregation-related epitopes formed by unrelated amyloid sequences. Understanding the mechanisms underlying these unusual properties and the structures of their corresponding epitopes is crucial for the understanding why antibodies display different therapeutic activities and for the development of more effective therapeutic agents. Here we employed a novel "epitomic" approach to map the fine structure of the epitopes of 28 monoclonal antibodies against amyloid-beta using immunoselection of random sequences from a phage display library, deep sequencing, and pattern analysis to define the critical sequence elements recognized by the antibodies. Although most of the antibodies map to major linear epitopes in the amino terminal 1 to 14 residues of Aß, the antibodies display differences in the target sequence residues that are critical for binding and in their individual preferences for nontarget residues, indicating that the antibodies bind to alternative conformations of the sequence by different mechanisms. Epitomic analysis also identifies discontinuous, nonoverlapping sequence Aß segments that may constitute the conformational epitopes that underlie the aggregation specificity of antibodies. Aggregation-specific antibodies recognize sequences that display a significantly higher predicted propensity for forming amyloid than antibodies that recognize the monomer, indicating that the ability of random sequences to aggregate into amyloid is a critical element of their binding mechanism.

Vaccines based on Plasmodium falciparum apical membrane antigen 1 (AMA1) have failed due to extensive polymorphism in AMA1. To assess the strain-specificity of antibody responses to malaria infection and AMA1 vaccination, we designed protein and peptide microarrays representing hundreds of unique AMA1 variants. Following clinical malaria episodes, children had short-lived, sequence-independent increases in average whole-protein seroreactivity, as well as strain-specific responses to peptides representing diverse epitopes. Vaccination resulted in dramatically increased seroreactivity to all 263 AMA1 whole-protein variants. High-density peptide analysis revealed that vaccinated children had increases in seroreactivity to four distinct epitopes that exceeded responses to natural infection. A single amino acid change was critical to seroreactivity to peptides in a region of AMA1 associated with strain-specific vaccine efficacy. Antibody measurements using whole antigens may be biased towards conserved, immunodominant epitopes. Peptide microarrays may help to identify immunogenic epitopes, define correlates of vaccine protection, and measure strain-specific vaccine-induced antibodies.

The repetitive interspersed family (RIFIN) and the subtelomeric variable open reading frame (STEVOR) family represent two of three major Plasmodium falciparum variant surface antigen families involved in malaria pathogenesis and immune evasion and are potential targets in the development of natural immunity. Protein and peptide microarrays populated with RIFINs and STEVORs associated with severe malaria vulnerability in Malian children were probed with adult and pediatric sera to identify epitopes that reflect malaria exposure. Adult sera recognized and reacted with greater intensity to all STEVOR proteins than pediatric sera did. Serorecognition of and seroreactivity to peptides within the semiconserved domain of STEVORs increased with age and seasonal malaria exposure, while serorecognition and seroreactivity increased for the semiconserved and second hypervariable domains of RIFINs only with age. Serologic responses to RIFIN and STEVOR peptides within the semiconserved domains may play a role in natural immunity to severe malaria.IMPORTANCE Malaria, an infectious disease caused by the parasite Plasmodium falciparum, causes nearly 435,000 deaths annually worldwide. RIFINs and STEVORs are two variant surface antigen families that are involved in malaria pathogenesis and immune evasion. Recent work has shown that a lack of humoral immunity to these proteins is associated with severe malaria vulnerability in Malian children. This is the first study to have compared serologic responses of children and adults to RIFINs and STEVORs in settings of malaria endemicity and to examine such serologic responses before and after a clinical malaria episode. Using microarrays, we determined that the semiconserved domains in these two parasite variant surface antigen families harbor peptides whose seroreactivity reflects malaria exposure. A similar approach has the potential to illuminate the role of variant surface antigens in the development of natural immunity to clinical malaria. Potential vaccines for severe malaria should include consideration of peptides within the semiconserved domains of RIFINs and STEVORs.

Immunization with sporozoites under chloroquine chemoprophylaxis (CPS) induces distinctly preerythrocytic and long-lasting sterile protection against homologous controlled human malaria infection (CHMI). To identify possible humoral immune correlates of protection, plasma samples were collected from 38 CPS-immunized Dutch volunteers for analysis using a whole Plasmodium falciparum proteome microarray with 7,455 full-length or segmented protein features displaying about 91% of the total P. falciparum proteome. We identified 548 reactive antigens representing 483 unique proteins. Using the breadth of antibody responses for each subject in a mixture-model algorithm, we observed a trimodal pattern, with distinct groups of 16 low responders, 19 medium responders, and 3 high responders. Fifteen out of 16 low responders, 12 of the 19 medium responders, and 3 out of 3 high responders were fully protected from a challenge infection. In the medium-responder group, we identified six novel antigens associated with protection (area under the curve [AUC] value of ≥0.75; P

Current seasonal influenza virus vaccines engender antibody-mediated protection that is hemagglutinin (HA) subtype specific and relatively short-lived. Coverage for other subtypes or even variants within a subtype could be improved from a better understanding of the factors that promote HA-specific antibody cross-reactivity. Current assays to evaluate cross-reactivity, such as the ELISA, require a separate test for each antigen and are neither high-throughput nor sample-sparing. To address this need, we produced an array of 283 purified HA proteins from influenza A virus subtypes H1 to H16 and H18 and influenza B virus. To evaluate performance, arrays were probed with sera from individuals before and after a booster dose of inactivated heterologous H5N1 vaccine and naturally infected cases at presentation and follow-up during the 2010 to 2011 influenza season, when H3N2 was prevalent. The response to the H5 vaccine boost was IgG only and confined to H5 variants. The response to natural H3N2 infection consisted of IgG and IgA and was reactive with all H3 variants displayed, as well as against other group 2 HA subtypes. In both groups, responses to HA1 proteins were subtype specific. In contrast, baseline signals were higher, and responses broader, against full-length HA proteins (HA1+HA2) compared to HA1 alone. We propose that these elevated baseline signals and breadth come from the recognition of conserved epitopes in the stalk domain by cross-reactive antibodies accumulated from previous exposure(s) to seasonal influenza virus. This array is a valuable high-throughput alternative to the ELISA for monitoring specificity and cross-reactivity of HA antibodies and has many applications in vaccine development.IMPORTANCE Seasonal influenza is a serious public health problem because the viral infection spreads easily from person to person and because of antigenic drift in neutralizing epitopes. Influenza vaccination is the most effective way to prevent the disease, although challenging because of the constant evolution of influenza virus subtypes. Our high-throughput protein microarrays allow for interrogation of subunit-specific IgG and IgA responses to 283 different HA proteins comprised of HA1 and HA2 domains as well as full-length HA proteins. This provides a tool that allows for novel insights into the response to exposure to influenza virus antigens. Data generated with our technology will enhance our understanding of the factors that improve the strength, breadth, and durability of vaccine-mediated immune responses and develop more effective vaccines.

Maternal antibodies may play a role in protecting newborns against malaria disease. Plasmodium falciparum parasite surface antigens are diverse, and protection from infection requires allele-specific immunity. Although malaria-specific antibodies have been shown to cross the placenta, the extent to which antibodies that respond to the full repertoire of diverse antigens are transferred from the mother to the infant has not been explored. Understanding the breadth of maternal antibody responses and to what extent these antibodies are transferred to the child can inform vaccine design and evaluation. We probed plasma from cord blood and serum from mothers at delivery using a customized protein microarray that included variants of malaria vaccine target antigens to assess the intensity and breadth of seroreactivity to three malaria vaccine candidate antigens in mother-newborn pairs in Malawi. Among the 33 paired specimens that were assessed, mothers and newborns had similar intensity and repertoire of seroreactivity. Maternal antibody levels against vaccine candidate antigens were the strongest predictors of infant antibody levels. Placental malaria did not significantly impair transplacental antibody transfer. However, mothers with placental malaria had significantly higher antibody levels against these blood-stage antigens than mothers without placental malaria. The repertoire and levels of infant antibodies against a wide range of malaria vaccine candidate antigen variants closely mirror maternal levels in breadth and magnitude regardless of evidence of placental malaria. Vaccinating mothers with an effective malaria vaccine during pregnancy may induce high and potentially protective antibody repertoires in newborns.

BackgroundCholera is a severe dehydrating illness of humans caused by toxigenic strains of Vibrio cholerae O1 or O139. Identification of immunogenic V. cholerae antigens could lead to a better understanding of protective immunity in human cholera.MethodsWe probed microarrays containing 3652 V. cholerae antigens with plasma and antibody-in-lymphocyte supernatant (ALS, a surrogate marker of mucosal immune responses) from patients with severe cholera caused by V. cholerae O1 in Bangladesh and age-, sex-, and ABO-matched Bangladeshi controls. We validated a subset of identified antigens using enzyme-linked immunosorbent assay.ResultsOverall, we identified 608 immunoreactive V. cholerae antigens in our screening, 59 of which had higher immunoreactivity in convalescent compared with acute-stage or healthy control samples (34 in plasma, 39 in mucosal ALS; 13 in both sample sets). Identified antigens included cholera toxin B and A subunits, V. cholerae O-specific polysaccharide and lipopolysaccharide, toxin coregulated pilus A, sialidase, hemolysin A, flagellins (FlaB, FlaC, and FlaD), phosphoenolpyruvate-protein phosphotransferase, and diaminobutyrate-2-oxoglutarate aminotransferase.ConclusionsThis study is the first antibody profiling of the mucosal and systemic antibody responses to the nearly complete V. cholerae O1 protein immunome; it has identified antigens that may aid in the development of an improved cholera vaccine.

The hallmark of vaccines is their ability to prevent the spread of infectious pathogens and thereby serve as invaluable public health tool. Despite their medical relevance, there is a gap in our understanding of the physiological factors that mediate innate and adaptive immune response to vaccines. The endocannabinoid (eCB) system is a critical modulator of homeostasis in vertebrates. Our results indicate that macrophages and dendritic cells produce the endocannabinoid, 2-arachidonoyl-sn-glycerol (2-AG) upon antigen activation. We have also established that 2-AG levels are upregulated in the serum and in the lymph node of mice during vaccination. We hypothesized that the intrinsic release of eCBs from immune cells during activation by pathogenic antigens mitigate inflammation, but also suppress overall innate and adaptive immune response. Here we demonstrate, for the first time, that transient administration of the cannabinoid receptor 2 antagonist AM630 (10 mg/kg) or inverse agonist JTE907 (3 mg/kg) during immunization heightens the intensity and breadth of antigen-specific immune responses in young and aged mice through the upregulation of immunomodulatory genes in secondary lymphoid tissues.

Improved serodiagnostic tests for typhoid fever (TF) are needed for surveillance, to facilitate patient management, curb antibiotic resistance, and inform public health programs. To address this need, IgA, IgM and IgG ELISAs using Salmonella enterica serovar Typhi (S. Typhi) lipopolysaccharide (LPS) and hemolysin E (t1477) protein were conducted on 86 Nigerian pediatric TF and 29 non-typhoidal Salmonella (NTS) cases, 178 culture-negative febrile cases, 28 "other" (i.e., non-Salmonella) pediatric infections, and 48 healthy Nigerian children. The best discrimination was achieved between TF and healthy children. LPS-specific IgA and IgM provided receiver operator characteristic areas under the curve (ROC AUC) values of 0.963 and 0.968, respectively, and 0.978 for IgA+M combined. Similar performance was achieved with t1477-specific IgA and IgM (0.968 and 0.968, respectively; 0.976 combined). IgG against LPS and t1477 was less accurate for discriminating these groups, possibly as a consequence of previous exposure, although ROC AUC values were still high (0.928 and 0.932, respectively). Importantly, discrimination between TF and children with other infections was maintained by LPS-specific IgA and IgM (AUC = 0.903 and 0.934, respectively; 0.938 combined), and slightly reduced for IgG (0.909), while t1477-specific IgG performed best (0.914). A similar pattern was seen when comparing TF with other infections from outside Nigeria. The t1477 may be recognized by cross-reactive antibodies from other acute infections, although a robust IgG response may provide some diagnostic utility in populations where incidence of other infections is low, such as in children. The data are consistent with IgA and IgM against S. Typhi LPS being specific markers of acute TF.

BackgroundLeptospirosis is an important zoonotic disease worldwide. Humans usually present a mild non-specific febrile illness, but a proportion of them develop more severe outcomes, such as multi-organ failure, lung hemorrhage and death. Such complications are thought to depend on several factors, including the host immunity. Protective immunity is associated with humoral immune response, but little is known about the immune response mounted during naturally-acquired Leptospira infection.Methods and principal findingsHere, we used protein microarray chip to profile the antibody responses of patients with severe and mild leptospirosis against the complete Leptospira interrogans serovar Copenhageni predicted ORFeome. We discovered a limited number of immunodominant antigens, with 36 antigens specific to patients, of which 11 were potential serodiagnostic antigens, identified at acute phase, and 33 were potential subunit vaccine targets, detected after recovery. Moreover, we found distinct antibody profiles in patients with different clinical outcomes: in the severe group, overall IgM responses do not change and IgG responses increase over time, while both IgM and IgG responses remain stable in the mild patient group. Analyses of individual patients' responses showed that >74% of patients in the severe group had significant IgG increases over time compared to 29% of patients in the mild group. Additionally, 90% of IgM responses did not change over time in the mild group, compared to ~51% in the severe group.ConclusionsIn the present study, we detected antibody profiles associated with disease severity and speculate that patients with mild disease were protected from severe outcomes due to pre-existing antibodies, while patients with severe leptospirosis demonstrated an antibody profile typical of first exposure. Our findings represent a significant advance in the understanding of the humoral immune response to Leptospira infection, and we have identified new targets for the development of subunit vaccines and diagnostic tests.

Inexpensive, easy-to-use, and highly sensitive diagnostic tests are currently unavailable for typhoid fever. To identify candidate serodiagnostic markers, we have probed microarrays displaying the full Salmonella enterica serovar Typhi (S. Typhi) proteome of 4,352 different proteins + lipopolysaccharides (LPSs), with sera from Nigerian pediatric typhoid and other febrile cases, Nigerian healthy controls, and healthy U.S. adults. Nigerian antibody profiles were broad (∼500 seropositive antigens) and mainly low level, with a small number of stronger "hits," whereas the profile in U.S. adults was < 1/5 as broad, consistent with endemic exposure in Nigeria. Nigerian profiles were largely unaffected by clinical diagnosis, although the response against t1477 (hemolysin E) consistently emerged as stronger in typhoid cases. The response to LPS was also a strong discriminator of healthy controls and typhoid, although LPS did not discriminate between typhoid and nontyphoidal Salmonella (NTS) disease. As a first step toward the development of a point-of-care diagnostic, t1477 and LPS were evaluated on immunostrips. Both provided good discrimination between healthy controls and typhoid/NTS disease. Such a test could provide a useful screen for salmonellosis (typhoid and NTS disease) in suspected pediatric cases that present with undefined febrile disease.

Little is known about the humoral immune response against DNA prime-recombinant adenovirus 5 (rAd5) boost HIV vaccine among HIV-infected patients on long-term suppressive antiretroviral therapy (ART). Previous studies emphasized cellular immune responses; however, current research suggests both cellular and humoral responses are likely required for a successful therapeutic vaccine. Thus, we aimed to understand antibody response and function induced by vaccination of ART-treated HIV-1-infected patients with immune recovery. All subjects participated in EraMune 02, an open-label randomized clinical trial of ART intensification followed by a six plasmid DNA prime (envA, envB, envC, gagB, polB, nefB) and rAd5 boost HIV vaccine with matching inserts. Antibody binding levels were determined with a recently developed microarray approach. We also analyzed neutralization efficiency and antibody-dependent cellular cytotoxicity (ADCC). We found that the DNA prime-rAd5 boost vaccine induced a significant cross-clade HIV-specific antibody response, which correlated with antibody neutralization efficiency. However, despite the increase in antibody binding levels, the vaccine did not significantly stimulate neutralization or ADCC responses. This finding was also reflected by a lack of change in total CD4+ cell associated HIV DNA in those who received the vaccine. Our results have important implications for further therapeutic vaccine design and administration, especially in HIV-1 infected patients, as boosting of preexisting antibody responses are unlikely to lead to clearance of latent proviruses in the HIV reservoir.

BackgroundMalaria is a public health problem in parts of Thailand, where Plasmodium falciparum and Plasmodium vivax are the main causes of infection. In the northwestern border province of Tak parasite prevalence is now estimated to be less than 1% by microscopy. Nonetheless, microscopy is insensitive at low-level parasitaemia. The objective of this study was to assess the current epidemiology of falciparum and vivax malaria in Tak using molecular methods to detect exposure to and infection with parasites; in particular, the prevalence of asymptomatic infections and infections with submicroscopic parasite levels.MethodsThree-hundred microlitres of whole blood from finger-prick were collected into capillary tubes from residents of a sentinel village and from patients at a malaria clinic. Pelleted cellular fractions were screened by quantitative PCR to determine parasite prevalence, while plasma was probed on a protein microarray displaying hundreds of P. falciparum and P. vivax proteins to obtain antibody response profiles in those individuals.ResultsOf 219 samples from the village, qPCR detected 25 (11.4%) Plasmodium sp. infections, of which 92% were asymptomatic and 100% were submicroscopic. Of 61 samples from the clinic patients, 27 (44.3%) were positive by qPCR, of which 25.9% had submicroscopic parasite levels. Cryptic mixed infections, misdiagnosed as single-species infections by microscopy, were found in 7 (25.9%) malaria patients. All sample donors, parasitaemic and non-parasitaemic alike, had serological evidence of parasite exposure, with 100% seropositivity to at least 54 antigens. Antigens significantly associated with asymptomatic infections were P. falciparum MSP2, DnaJ protein, putative E1E2 ATPase, and three others.ConclusionThese findings suggest that parasite prevalence is higher than currently estimated by local authorities based on the standard light microscopy. As transmission levels drop in Thailand, it may be necessary to employ higher throughput and sensitivity methods for parasite detection in the phase of malaria elimination.

Malaria elimination efforts would benefit from vaccines that block transmission of Plasmodium falciparum gametocytes from humans to mosquitoes. A clear understanding of gametocyte-specific antibody responses in exposed populations could help determine whether transmission-blocking vaccines (TBV) would be boosted by natural gametocyte exposure, and also inform the development of serologic tools to monitor gametocyte exposure in populations targeted for malaria elimination. To this end, plasma was collected from Malian children and adults before and after the 6-month malaria season and probed against a microarray containing 1,204 P. falciparum proteins. Using publicly available proteomic data, we classified 91 proteins as gametocyte specific and 69 as proteins not expressed by gametocytes. The overall breadth and magnitude of gametocyte-specific IgG responses increased during the malaria season, although they were consistently lower than IgG responses to nongametocyte antigens. Notably, IgG specific for the TBV candidates Pfs48/45 and Pfs230 increased during the malaria season. In addition, IgGs specific for the gametocyte proteins Pfmdv1, Pfs16, PF3D7_1346400, and PF3D7_1024800 were detected in nearly all subjects, suggesting that seroconversion to these proteins may be a sensitive indicator of gametocyte exposure, although further studies are needed to determine the specificity and kinetics of these potential serologic markers. These findings suggest that TBV-induced immunity would be boosted through natural gametocyte exposure, and that antibody responses to particular antigens may reliably indicate gametocyte exposure.

Tools to reliably measure Plasmodium falciparum (Pf) exposure in individuals and communities are needed to guide and evaluate malaria control interventions. Serologic assays can potentially produce precise exposure estimates at low cost; however, current approaches based on responses to a few characterized antigens are not designed to estimate exposure in individuals. Pf-specific antibody responses differ by antigen, suggesting that selection of antigens with defined kinetic profiles will improve estimates of Pf exposure. To identify novel serologic biomarkers of malaria exposure, we evaluated responses to 856 Pf antigens by protein microarray in 186 Ugandan children, for whom detailed Pf exposure data were available. Using data-adaptive statistical methods, we identified combinations of antibody responses that maximized information on an individual's recent exposure. Responses to three novel Pf antigens accurately classified whether an individual had been infected within the last 30, 90, or 365 d (cross-validated area under the curve = 0.86-0.93), whereas responses to six antigens accurately estimated an individual's malaria incidence in the prior year. Cross-validated incidence predictions for individuals in different communities provided accurate stratification of exposure between populations and suggest that precise estimates of community exposure can be obtained from sampling a small subset of that community. In addition, serologic incidence predictions from cross-sectional samples characterized heterogeneity within a community similarly to 1 y of continuous passive surveillance. Development of simple ELISA-based assays derived from the successful selection strategy outlined here offers the potential to generate rich epidemiologic surveillance data that will be widely accessible to malaria control programs.

Acquisition of acute toxoplasmosis during the first trimester of pregnancy can have catastrophic consequences for the foetus. Diagnosis is routinely based on the detection of maternal Toxoplasma gondii--antibodies using whole parasite extracts as detection antigen. While such assays are sensitive, they show no specificity for the stage of infection. We hypothesized diagnosis might be improved using recombinant antigens for detection, particularly if antibodies to certain antigen(s) were associated with early or late stages of infection. To address this, protein microarrays comprising 1513 T. gondii exon products were probed with well-characterized sera from seronegative ('N') controls, and acute ('A'), chronic/IgM-persisting ('C/M') and chronic ('C') toxoplasmosis cases from Turkey. Three reactive exon products recognized preferentially in A infections, and three recognized preferentially in C/M infections, were expressed in Escherichia coli and tested for discrimination in IgG ELISAs. The best discriminators were exon 1 of TGME49_086450 (GRA5) which detected C/M infections with 70.6% sensitivity and 81.8% specificity, and exon 6 of TGME49_095700 (ubiquitin transferase domain-containing protein) which detected A infections with 84.8% sensitivity and 82.4% specificity. Overall, the data support a recombinant protein approach for the development of improved serodiagnostic tests for toxoplasmosis.

In recent years, high throughput discovery of human recombinant monoclonal antibodies (mAbs) has been applied to greatly advance our understanding of the specificity, and functional activity of antibodies against HIV. Thousands of antibodies have been generated and screened in functional neutralization assays, and antibodies associated with cross-strain neutralization and passive protection in primates, have been identified. To facilitate this type of discovery, a high throughput-screening tool is needed to accurately classify mAbs, and their antigen targets. In this study, we analyzed and evaluated a prototype microarray chip comprised of the HIV-1 recombinant proteins gp140, gp120, gp41, and several membrane proximal external region peptides. The protein microarray analysis of 11 HIV-1 envelope-specific mAbs revealed diverse binding affinities and specificities across clades. Half maximal effective concentrations, generated by our chip analysis, correlated significantly (P

The cervix and/or fallopian tubes of pigtailed macaques were experimentally infected with Chlamydia trachomatis. Their sera were collected at varying time points and screened for identification of immunodominant antigens using a whole-genome protein microarray. The effect of doxycycline treatment on the antibody response generated in these macaques was also investigated. Twenty-five female macaques were infected with C. trachomatis serovars D or E in the cervix and/or fallopian tubes. Bloods were collected at baseline and at various intervals after challenge. Serum samples were tested for antibodies using a C. trachomatis serovar D protein microarray. Twenty chlamydial antigens reacted with sera from at least 68% (17/25) of the macaques. In addition to some well-known chlamydial antigens, nine different proteins, not previously recognized as immunodominant, including four hypothetical proteins (CT005, CT066, CT360 and CT578), were identified. Monkeys infected in the fallopian tubes developed a more robust antibody response than animals inoculated in the cervix. Treatment with doxycycline significantly decreased Chlamydia-specific antibody levels. In summary, using protein microarray serum samples from experimentally infected pigtailed macaques were screened for immunodominant chlamydial antigens. These antigens can now be tested in animal models for their ability to protect and as markers of disease progression.Biological significanceThis is the first time that Chlamydia trachomatis immunodominant antigens have been identified in pigtailed macaques following a uterine cervix or a fallopian tubes infection. These immunodominant antigens can now be used to vaccinate non-human primates and determine their ability to protect against a C. trachomatis genital infection. Proteins that are protective can subsequently be tested in humans. Amongst the immunodominant antigens some were predominantly recognized by sera from macaques inoculated in the fallopian tubes rather than in the cervix and therefore, may be markers for upper genital tract pathology. In addition, treatment with doxycycline following infection significantly decreased Chlamydia-specific antibody levels. This information can be used to evaluate the efficacy of antibiotic treatment and potentially susceptibility to reinfection.

A complete understanding of the factors that determine selection of antigens recognized by the humoral immune response following infectious agent challenge is lacking. Here we illustrate a systems biology approach to identify the antibody signature associated with Brucella melitensis (Bm) infection in humans and predict proteomic features of serodiagnostic antigens. By taking advantage of a full proteome microarray expressing previously cloned 1406 and newly cloned 1640 Bm genes, we were able to identify 122 immunodominant antigens and 33 serodiagnostic antigens. The reactive antigens were then classified according to annotated functional features (COGs), computationally predicted features (e.g., subcellular localization, physical properties), and protein expression estimated by mass spectrometry (MS). Enrichment analyses indicated that membrane association and secretion were significant enriching features of the reactive antigens, as were proteins predicted to have a signal peptide, a single transmembrane domain, and outer membrane or periplasmic location. These features accounted for 67% of the serodiagnostic antigens. An overlay of the seroreactive antigen set with proteomic data sets generated by MS identified an additional 24%, suggesting that protein expression in bacteria is an additional determinant in the induction of Brucella-specific antibodies. This analysis indicates that one-third of the proteome contains enriching features that account for 91% of the antigens recognized, and after B. melitensis infection the immune system develops significant antibody titers against 10% of the proteins with these enriching features. This systems biology approach provides an empirical basis for understanding the breadth and specificity of the immune response to B. melitensis and a new framework for comparing the humoral responses against other microorganisms.

Background: Acquisition of malaria immunity in low transmission areas usually occurs after relatively few exposures to the parasite. A recent Plasmodium vivax experimental challenge trial in malaria naïve and semi-immune volunteers from Colombia showed that all naïve individuals developed malaria symptoms, whereas semi-immune subjects were asymptomatic or displayed attenuated symptoms. Sera from these individuals were analyzed by protein microarray to identify antibodies associated with clinical protection. Methodology/Principal Findings: Serum samples from naïve (n = 7) and semi-immune (n = 9) volunteers exposed to P. vivax sporozoite-infected mosquito bites were probed against a custom protein microarray displaying 515 P. vivax antigens. The array revealed higher serological responses in semi-immune individuals before the challenge, although malaria naïve individuals also had pre-existing antibodies, which were higher in Colombians than US adults (control group). In both experimental groups the response to the P. vivax challenge peaked at day 45 and returned to near baseline at day 145. Additional analysis indicated that semi-immune volunteers without fever displayed a lower response to the challenge, but recognized new antigens afterwards. Conclusion: Clinical protection against experimental challenge in volunteers with previous P. vivax exposure was associated with elevated pre-existing antibodies, an attenuated serological response to the challenge and reactivity to new antigens. [ABSTRACT FROM AUTHOR]

Background. People of the Fulani ethnic group are more resistant to malaria compared with genetically distinct ethnic groups, such as the Dogon people, in West Africa, and studies suggest that this resistance is mediated by enhanced antibody responses to Plasmodium falciparum antigens. However, prior studies measured antibody responses to <0.1% of P falciparum proteins, so whether the Fulani mount an enhanced and broadly reactive immunoglobulin (Ig)M and IgG response to P falciparum remains unknown. In general, little is known about the extent to which host genetics influence the overall antigen specificity of IgM and IgG responses to natural infections. Methods. In a cross-sectional study in Mali, we collected plasma from asymptomatic, age-matched Fulani (n = 24) and Dogon (n = 22) adults with or without concurrent P falciparum infection. We probed plasma against a protein microarray containing 1087 P falciparum antigens and compared IgM and IgG profiles by ethnicity. Results. We found that the breadth and magnitude of P falciparum-specific IgM and IgG responses were significantly higher in the malaria-resistant Fulani versus the malaria-susceptible Dogon, and, unexpectedly, P falciparum-specific IgM responses more strongly distinguished the 2 ethnic groups. Conclusions. These findings point to an underappreciated role for IgM in protection from malaria, and they suggest that host genetics may influence the antigen specificity of IgM and IgG responses to infection. [ABSTRACT FROM AUTHOR]

Extreme diversity of the major Plasmodium falciparum antigen, PfEMP1, poses a barrier to identifying targets of immunity to malaria. Here, we used protein microarrays containing hundreds of variants of the DBLα domain of PfEMP1 to cover the diversity of Papua New Guinean (PNG) parasites. Probing the plasma of a longitudinal cohort of malaria-exposed PNG children showed that group 2 DBLα antibodies were moderately associated with a lower risk of uncomplicated malaria, whereas individual variants were only weakly associated with clinical immunity. In contrast, antibodies to 85 individual group 1 and 2 DBLα variants were associated with a 70%–100% reduction in severe malaria. Of these, 17 variants were strong predictors of severe malaria. Analysis of full-length PfEMP1 sequences from PNG samples shows that these 17 variants are linked to pathogenic CIDR domains. This suggests that whereas immunity to uncomplicated malaria requires a broad repertoire of antibodies, immunity to severe malaria targets a subset of conserved variants. These findings provide insights into antimalarial immunity and potential antibody biomarkers for disease risk. • Group 1 and 2 DBLα domains are serodominant in PNG children exposed to malaria • Antibodies against individual DBLα are weakly associated with risk of clinical malaria • Antibodies against specific conserved DBLα are strongly associated with severe malaria • Protective DBLα antibodies are potential biomarkers for severe disease risk Identifying targets of antimalarial immunity is essential for vaccine development but compromised by the enormous diversity of malaria parasite surface antigens. Tessema et al. show that severe malaria immunity targets a subset of antigenically conserved parasite proteins, whereas clinical immunity requires a broad repertoire of antibodies to diverse proteins. [ABSTRACT FROM AUTHOR]

Abstract: Tularemia is caused by the Gram-negative facultative intracellular bacterium Francisella tularensis, which has been classified as a category A select agent—a likely bioweapon. The high virulence of F. tularensis and the threat of engineered antibiotic resistant variants warrant the development of new therapies to combat this disease. We have characterized 14 anti-Francisella hybridoma antibodies derived from mice infected with F. tularensis live vaccine strain (LVS) for potential use as immunotherapy of tularemia. All 14 antibodies cross-reacted with virulent F. tularensis type A clinical isolates, 8 bound to a purified preparation of LVS LPS, and 6 bound to five protein antigens, identified by proteome microarray analysis. An IgG2a antibody, reactive with the LPS preparation, conferred full protection when administered either systemically or intranasally to BALB/c mice post challenge with a lethal dose of intranasal LVS; three other antibodies prolonged survival. These anti-Francisella hybridoma antibodies could be converted to chimeric versions with mouse V regions and human C regions to serve as components of a recombinant polyclonal antibody for clinical testing as immunotherapy of tularemia. The current study is the first to employ proteome microarrays to identify the target antigens of anti-Francisella monoclonal antibodies and the first to demonstrate the systemic and intranasal efficacy of monoclonal antibodies for post-exposure treatment of respiratory tularemia. [Copyright &y& Elsevier]