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Children with hemoglobin AC or AS have decreased susceptibility to clinical malaria. Parasite variant surface antigen (VSA) presentation on the surface of infected erythrocytes is altered in erythrocytes with hemoglobin C (Hb AC) or sickle trait (Hb AS) mutations in vitro. The protective role of incomplete or altered VSA presentation against clinical malaria in individuals with Hb AC or AS is unclear. Using a high-throughput protein microarray, we sought to use serological responses to VSAs as a measure of host exposure to VSAs among Malian children with Hb AC, Hb AS, or wildtype hemoglobin (Hb AA). In uncomplicated malaria, when compared to Hb AA children, Hb AC children had significantly lower serological responses to extracellular Plasmodium falciparum erythrocyte membrane protein-1 (PfEMP1) domains but did not differ in responses to intracellular PfEMP1 domains and other VSAs, including members of the repetitive interspersed family (RIFIN) and subtelomeric variable open reading frame (STEVOR) family. Healthy children with Hb AC and Hb AS genotypes recognized fewer extracellular PfEMP1s compared to children with Hb AA, especially CD36-binding PfEMP1s. These reduced serologic responses may reflect reduced VSA presentation or lower parasite exposure in children with Hb AC or AS and provide insights into mechanisms of protection., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: PfSPZ Vaccine, a promising pre-erythrocytic stage malaria vaccine candidate based on whole, radiation-attenuated Plasmodium falciparum (Pf) sporozoites (SPZ), has proven safe and effective in mediating sterile protection from malaria in malaria-naïve and exposed healthy adults. Vaccine-induced protection presumably depends on cellular responses to early parasite liver stages, but humoral immunity contributes., Methods: On custom-made Pf protein microarrays, we profiled IgG and IgM responses to PfSPZ Vaccine and subsequent homologous controlled human malaria infection (CHMI) in 21 Tanzanian adults with (n = 12) or without (n = 9) HIV infection. Expression of the main identified immunogens in the pre-erythrocytic parasite stage was verified by immunofluorescence detection using freshly purified PfSPZ and an in vitro model of primary human hepatocytes., Findings: Independent of HIV infection status, immunisation induced focused IgG and IgM responses to circumsporozoite surface protein (PfCSP) and merozoite surface protein 5 (PfMSP5). We show that PfMSP5 is detectable on the surface and in the apical complex of PfSPZ., Interpretation: Our data demonstrate that HIV infection does not affect the quantity of the total IgG and IgM antibody responses to PfCSP and PfMSP5 after immunization with PfSPZ Vaccine. PfMSP5 represents a highly immunogenic, so far underexplored, target for vaccine-induced antibodies in malaria pre-exposed volunteers., Funding: This work was supported by the Equatorial Guinea Malaria Vaccine Initiative (EGMVI), the Clinical Trial Platform of the German Center for Infection Research (TTU 03.702), the Swiss Government Excellence Scholarships for Foreign Scholars and Artists (grant 2016.0056) and the Interdisciplinary Center for Clinical Research doctoral program of the Tübingen University Hospital. The funders had no role in design, analysis, or reporting of this study., Competing Interests: Declaration of interests Sanaria Inc. manufactured PfSPZ Vaccine and PfSPZ Challenge and was the sponsor of the clinical trial. YA, BKLS, TLR, TM, and SLH are salaried, full-time employees of Sanaria, and LWPC and PFB were full-time, salaried employees of Sanaria at the time the trial was conducted. All authors associated with Sanaria have potential conflicts of interest. All other authors declare that they have no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Immunization through repeated direct venous inoculation of Plasmodium falciparum (Pf) sporozoites (PfSPZ) under chloroquine chemoprophylaxis, using the PfSPZ Chemoprophylaxis Vaccine (PfSPZ-CVac), induces high-level protection against controlled human malaria infection (CHMI). Humoral and cellular immunity contribute to vaccine efficacy but only limited information about the implicated Pf-specific antigens is available. Here, we examined Pf-specific antibody profiles, measured by protein arrays representing the full Pf proteome, of 40 placebo- and PfSPZ-immunized malaria-naïve volunteers from an earlier published PfSPZ-CVac dose-escalation trial. For this purpose, we both utilized and adapted supervised machine learning methods to identify predictive antibody profiles at two different time points: after immunization and before CHMI. We developed an adapted multitask support vector machine (SVM) approach and compared it to standard methods, i.e. single-task SVM, regularized logistic regression and random forests. Our results show, that the multitask SVM approach improved the classification performance to discriminate the protection status based on the underlying antibody-profiles while combining time- and dose-dependent data in the prediction model. Additionally, we developed the new fEature diStance exPlainabilitY (ESPY) method to quantify the impact of single antigens on the non-linear multitask SVM model and make it more interpretable. In conclusion, our multitask SVM model outperforms the studied standard approaches in regard of classification performance. Moreover, with our new explanation method ESPY, we were able to interpret the impact of Pf-specific antigen antibody responses that predict sterile protective immunity against CHMI after immunization. The identified Pf-specific antigens may contribute to a better understanding of immunity against human malaria and may foster vaccine development., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: J.J.C. is a salaried employee of Antigen Discovery Inc., the manufacturer of the protein microarrays and performer of the experiments from which the data was derived. S.L.H. is an owner and salaried employee of Sanaria Inc. the manufacturer of PfSPZ-CVac and the Sponsor of the clinical trial from which the data was derived. P.L.F. is founder and stock holder of Nanommune Inc. and Antigen Discovery Inc., (Copyright: © 2024 Wistuba-Hamprecht et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Malaria sterile immunity has been reproducibly induced by immunization with Plasmodium radiation-attenuated sporozoites (RAS). Analyses of sera from RAS-immunized individuals allowed the identification of P. falciparum antigens, such as the circumsporozoite protein (CSP), the basis for the RTS, S and R21Matrix-M vaccines. Similar advances in P. vivax (Pv) vaccination have been elusive. We previously reported 42% (5/12) of sterile protection in malaria-unexposed, Duffy-positive (Fy +) volunteers immunized with PvRAS followed by a controlled human malaria infection (CHMI). Using a custom protein microarray displaying 515 Pv antigens, we found a significantly higher reactivity to PvCSP and one hypothetical protein (PVX_089630) in volunteers protected against P. vivax infection. In mock-vaccinated Fy + volunteers, a strong antibody response to CHMI was also observed. Although the Fy- volunteers immunized with non-irradiated Pv-infected mosquitoes (live sporozoites) did not develop malaria after CHMI, they recognized a high number of antigens, indicating the temporary presence of asexual parasites in peripheral blood. Together, our findings contribute to the understanding of the antibody response to P. vivax infection and allow the identification of novel parasite antigens as vaccine candidates.Trial registration: ClinicalTrials.gov number: NCT01082341., (© 2024. The Author(s).)

Antibody responses to variant surface antigens (VSAs) produced by the malaria parasite Plasmodium falciparum may contribute to age-related natural immunity to severe malaria. One VSA family, P. falciparum erythrocyte membrane protein-1 (PfEMP1), includes a subset of proteins that binds endothelial protein C receptor (EPCR) in human hosts and potentially disrupts the regulation of inflammatory responses, which may lead to the development of severe malaria. We probed peptide microarrays containing segments spanning five PfEMP1 EPCR-binding domain variants with sera from 10 Malian adults and 10 children to determine the differences between adult and pediatric immune responses. We defined serorecognized peptides and amino acid residues as those that elicited a significantly higher antibody response than malaria-naïve controls. We aimed to identify regions consistently serorecognized among adults but not among children across PfEMP1 variants, potentially indicating regions that drive the development of immunity to severe malaria. Adult sera consistently demonstrated broader and more intense serologic responses to constitutive PfEMP1 peptides than pediatric sera, including peptides in EPCR-binding domains. Both adults and children serorecognized a significantly higher proportion of EPCR-binding peptides than peptides that do not directly participate in receptor binding, indicating a preferential development of serologic responses at functional residues. Over the course of a single malaria transmission season, pediatric serological responses increased between the start and the peak of the season, but waned as the transmission season ended. IMPORTANCE Severe malaria and death related to malaria disproportionately affect sub-Saharan children under 5 years of age, commonly manifesting as cerebral malaria and/or severe malarial anemia. In contrast, adults in malaria-endemic regions tend to experience asymptomatic or mild disease. Our findings indicate that natural immunity to malaria targets specific regions within the EPCR-binding domain, particularly peptides containing EPCR-binding residues. Epitopes containing these residues may be promising targets for vaccines or therapeutics directed against severe malaria. Our approach provides insight into the development of natural immunity to a binding target linked to severe malaria by characterizing an "adult-like" response as recognizing a proportion of epitopes within the PfEMP1 protein, particularly regions that mediate EPCR binding. This "adult-like" response likely requires multiple years of malaria exposure, as increases in pediatric serologic response over a single malaria transmission season do not appear significant., Competing Interests: The authors declare no conflict of interest.

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 .

Background: Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2)-infected patients exhibit disease ranging from asymptomatic to severe pneumonia, multi-organ failure, and death. convalescent COVID plasma (CCP) from recovered patients with high levels of neutralizing antibodies has demonstrated therapeutic efficacy to reduce the morbidity of coronavirus disease 2019 (COVID-19) in some studies. The development of assays to characterize the activity of CCP to neutralize SARS-CoV-2 infectivity offers the possibility to improve potential therapeutic efficacy. Lyophilization of CCP may increase the availability of this therapy. We hypothesized that SARS-CoV-2 antibody profiles of pooled lyophilized pathogen-reduced CCP from COVID-19-recovered blood donors retains virus-neutralizing efficacy as reported for frozen pathogen-reduced CCP., Methods: Pooled lyophilized pathogen-reduced plasma was prepared from recovered COVID plasma donors. Antibodies to SARS-CoV-2 were characterized in each donor plasma prior to pathogen reduction and lyophilization and after lyophilization of individual CCP, and in the lyophilized CCP pool. Several complimentary assays were used to characterize antibody levels, neutralizing capacity, and the spectrum of antigen reactivity. The mean values for individual plasma samples and the value in the pool were compared., Results: The mean ratio for antibody binding to SARS-CoV-2 antigens before and after treatment was 0.95 ± 0.22 mean fluorescent intensity (MFI) units. Antibody activity to an array of influenza virus antigens demonstrated a mean activity ratio of 0.92 ± 0.12 MFI before and after treatment., Conclusions: The antibody activity in pooled pathogen-reduced lyophilized CCPs demonstrated minimal impact due to pathogen reduction treatment and lyophilization., (© 2023 AABB.)

Vaccines are among the most cost-effective public health measures for controlling infectious diseases. Coxiella burnetii is the etiological agent of Q fever, a disease with a wide clinical spectrum that ranges from mild symptoms, such as fever and fatigue, to more severe disease, such as pneumonia and endocarditis. The formalin-inactivated whole-cell vaccine Q-VAX ® contains hundreds of antigens and confers lifelong protection in humans, but prior sensitization from infection or vaccination can result in deleterious reactogenic responses to vaccination. Consequently, there is great interest in developing non-reactogenic alternatives based on adjuvanted recombinant proteins. In this study, we aimed to develop a multivalent vaccine that conferred protection with reduced reactogenicity. We hypothesized that a multivalent vaccine consisting of multiple antigens would be more immunogenic and protective than a monovalent vaccine owing to the large number of potential protective antigens in the C. burnetii proteome. To address this, we identified immunogenic T and B cell antigens, and selected proteins were purified to evaluate with a combination adjuvant (IVAX-1), with or without C. burnetii lipopolysaccharide (LPS) in immunogenicity studies in vivo in mice and in a Hartley guinea pig intratracheal aerosol challenge model using C. burnetii strain NMI RSA 493. The data showed that multivalent vaccines are more immunogenic than monovalent vaccines and more closely emulate the protection achieved by Q-VAX. Although six antigens were the most immunogenic, we also discovered that multiplexing beyond four antigens introduces detectable reactogenicity, indicating that there is an upper limit to the number of antigens that can be safely included in a multivalent Q-fever vaccine. C. burnetii LPS also demonstrates efficacy as a vaccine antigen in conferring protection in an otherwise monovalent vaccine formulation, suggesting that its addition in multivalent vaccines, as demonstrated by a quadrivalent formulation, would improve protective responses., Competing Interests: SJ, JF, JH-D, LL, RN, AJas, AJai, PF, and DHD own shares in MyImmunome Inc. MyImmunome does not sell any products described in this paper, nor funded any part of the work described herein. Neither MyImmunome or its shareholders are likely to benefit from the results described in this publication. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Jan, Fratzke, Felgner, Hernandez-Davies, Liang, Nakajima, Jasinskas, Supnet, Jain, Felgner, Davies and Gregory.)

High systemic doses of adeno-associated viruses (AAVs) have been associated with immune-related serious adverse events (SAEs). Although AAV was well tolerated in preclinical models, SAEs were observed in clinical trials, indicating the need for improved preclinical models to understand AAV-induced immune responses. Here, we show that mice dual-dosed with AAV9 at 4-week intervals better recapitulate aspects of human immunity to AAV. In the model, anti-AAV9 immunoglobulin G (IgGs) increased in a linear fashion between the first and second AAV administrations. Complement activation was only observed in the presence of high levels of both AAV and anti-AAV IgG. Myeloid-derived pro-inflammatory cytokines were significantly induced in the same pattern as complement activation, suggesting that myeloid cell activation to AAV may rely on the presence of both AAV and anti-AAV IgG complexes. Single-cell RNA sequencing of peripheral blood mononuclear cells confirmed that activated monocytes were a primary source of pro-inflammatory cytokines and chemokines, which were significantly increased after a second AAV9 exposure. The same activated monocyte clusters expressed both Fcγ and complement receptors, suggesting that anti-AAV-mediated activation of myeloid cells through Fcγ receptors and/or complement receptors is one mechanism by which anti-AAV antigen complexes may prime antigen-presenting cells and amplify downstream immunity., Competing Interests: M.J.S., A.D.P., and C.S.Y. are co-founders of MyoGene Bio, a startup spun out of UCLA developing gene editing therapies for Duchenne muscular dystrophy., (© 2023 The Authors.)

Introduction: In 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., Methods: To 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., Results: The 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., Discussion: In 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., Competing Interests: The coronavirus antigen microarray is intellectual property of the Regents of the University of California that is licensed for commercialization to Nanommune Inc. Irvine, CA, a private company for which PF is the largest shareholder and several co-authors RA, AaJ, RN, and SK also own shares. Nanommune Inc. has a business partnership with Sino Biological Inc. Beijing, China which expressed and purified the antigens used in this study. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Hosseinian, de Assis, Khalil, Luu, Jain, Horvath, Nakajima, Palma, Hoang, Razzak, Garcia, Alger, Kalantari, Silzel, Jasinskas, Zaldivar, Schubl, Felgner and Khan.)

Background: While 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., Methods: We 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., Results: Among 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)., Conclusion: SARS-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., (© 2023. The Author(s).)

Background: Efficacy of donated COVID-19 convalescent plasma (dCCP) is uncertain and may depend on antibody titers, neutralizing capacity, timing of administration, and patient characteristics., Study Design and Methods: In a single-center hypothesis-generating prospective case-control study with 1:2 matched dCCP recipients to controls according to disease severity at day 1, hospitalized adults with COVID-19 pneumonia received 2 × 200 ml pathogen-reduced treated dCCP from 2 different donors. We evaluated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies in COVID-19 convalescent plasma donors and recipients using multiple antibody assays including a Coronavirus antigen microarray (COVAM), and binding and neutralizing antibody assays. Outcomes were dCCP characteristics, antibody responses, 28-day mortality, and dCCP -related adverse events in recipients., Results: Eleven of 13 dCCPs (85%) contained neutralizing antibodies (nAb). PRT did not affect dCCP antibody activity. Fifteen CCP recipients and 30 controls (median age 64 and 65 years, respectively) were enrolled. dCCP recipients received 2 dCCPs from 2 different donors after a median of one hospital day and 11 days after symptom onset. One dCCP recipient (6.7%) and 6 controls (20%) died (p = 0.233). We observed no dCCP-related adverse events. Transfusion of unselected dCCP led to heterogeneous SARS CoV-2 antibody responses. COVAM clustered dCCPs in 4 distinct groups and showed endogenous immune responses to SARS-CoV-2 antigens over 14-21 days post dCCP in all except 4 immunosuppressed recipients., Discussion: PRT did not impact dCCP anti-virus neutralizing activity. Transfusion of unselected dCCP did not impact survival and had no adverse effects. Variable dCCP antibodies and post-transfusion antibody responses indicate the need for controlled trials using well-characterized dCCP with informative assays., (© 2022 The Authors. Transfusion published by Wiley Periodicals LLC on behalf of AABB.)

High-throughput and rapid screening testing is highly desirable to effectively combat the rapidly evolving COVID-19 pandemic co-presents with influenza and seasonal common cold epidemics. Here, we present a general workflow for iterative development and validation of an antibody-based microarray assay for the detection of a respiratory viral panel: (a) antibody screening to quickly identify optimal reagents and assay conditions, (b) immunofluorescence assay design including signal amplification for low viral titers, (c) assay characterization with recombinant proteins, inactivated viral samples and clinical samples, and (d) multiplexing to detect a panel of common respiratory viruses. Using RT-PCR-confirmed SARS-CoV-2 positive and negative pharyngeal swab samples, we demonstrated that the antibody microarray assay exhibited a clinical sensitivity and specificity of 77.2% and 100%, respectively, which are comparable to existing FDA-authorized antigen tests. Moreover, the microarray assay is correlated with RT-PCR cycle threshold (Ct) values and is particularly effective in identifying high viral titers. The multiplexed assay can selectively detect SARS-CoV-2 and influenza virus, which can be used to discriminate these viral infections that share similar symptoms. Such protein microarray technology is amenable for scale-up and automation and can be broadly applied as a both diagnostic and research tool.

Current seasonal and pre-pandemic influenza vaccines induce short-lived predominantly strain-specific and limited heterosubtypic responses. To better understand how vaccine adjuvants AS03 and MF59 may provide improved antibody responses to vaccination, we interrogated serum from subjects who received 2 doses of inactivated monovalent influenza A/Indonesia/05/2005 vaccine with or without AS03 or MF59 using hemagglutinin (HA) microarrays (NCT01317758 and NCT01317745). The arrays were designed to reflect both full-length and globular head HA derived from 17 influenza A subtypes (H1 to H16 and H18) and influenza B strains. We observed significantly increased strain-specific and broad homo- and heterosubtypic antibody responses with both AS03 and MF59 adjuvanted vaccination with AS03 achieving a higher titer and breadth of IgG responses relative to MF59. The adjuvanted vaccine was also associated with the elicitation of stalk-directed antibody. We established good correlation of the array antibody responses to H5 antigens with standard HA inhibition and microneutralization titers., (© 2022. The Author(s).)

We used a protein microarray featuring Plasmodium falciparum field variants of a merozoite surface antigen to examine malaria exposure in Malian children with different severe malaria syndromes. Unlike children with cerebral malaria alone or severe malarial anemia alone, those with concurrent cerebral malaria and severe malarial anemia had serologic responses demonstrating a broader prior parasite exposure pattern than matched controls with uncomplicated disease. Comparison of levels of malaria-related cytokines revealed that children with the concurrent phenotype had elevated levels of interleukin (IL)-6, IL-8, and IL-10. Our results suggest that the pathophysiology of this severe subtype is unique and merits further investigation.

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., (© 2022. The Author(s).)

Sustained signaling through the B cell antigen receptor (BCR) is thought to occur only when antigen(s) crosslink or disperse multiple BCR units, such as by multimeric antigens found on the surfaces of viruses or bacteria. B cell-intrinsic Toll-like receptor (TLR) signaling synergizes with the BCR to induce and shape antibody production, hallmarked by immunoglobulin (Ig) class switch recombination (CSR) of constant heavy chains from IgM/IgD to IgG, IgA or IgE isotypes, and somatic hypermutation (SHM) of variable heavy and light chains. Full B cell differentiation is essential for protective immunity, where class switched high affinity antibodies neutralize present pathogens, memory B cells are held in reserve for future encounters, and activated B cells also serve as semi-professional APCs for T cells. But the rules that fine-tune B cell differentiation remain partially understood, despite their being essential for naturally acquired immunity and for guiding vaccine development. To address this in part, we have developed a cell culture system using splenic B cells from naive mice stimulated with several biotinylated ligands and antibodies crosslinked by streptavidin reagents. In particular, biotinylated lipopolysaccharide (LPS), a Toll-like receptor 4 (TLR4) agonist, and biotinylated anti-IgM were pre-assembled (multimerized) using streptavidin, or immobilized on nanoparticles coated with streptavidin, and used to active B cells in this precisely controlled, high throughput assay. Using B cell proliferation and Ig class switching as metrics for successful B cell activation, we show that the stimuli are both synergistic and dose-dependent. Crucially, the multimerized immunoconjugates are most active over a narrow concentration range. These data suggest that multimericity is an essential requirement for B cell BCR/TLRs ligands, and clarify basic rules for B cell activation. Such studies highlight the importance in determining the choice of single vs multimeric formats of antigen and PAMP agonists during vaccine design and development., Competing Interests: All of the authors own shares in Nanommune Inc. Nanommune does not sell any products described in this paper, nor funded any part of the work described herein. Neither Nanommune nor its shareholders are likely to benefit from the results described in this publication., (Copyright © 2022 Pone, Hernandez-Davies, Jan, Silzel, Felgner and Davies.)

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., Competing Interests: The authors have declared that no competing interests exist.

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., Competing Interests: The coronavirus antigen microarray is intellectual property of the Regents of the University of California that is licensed for commercialization to Nanommune Inc. (Irvine, CA), a private company for which PF is the largest shareholder and several co-authors (RA, AaJ, RN, and SK) also own shares. Nanommune Inc. has a business partnership with Sino Biological Inc. (Beijing, China) which expressed and purified the antigens used in this study. KP is invested in mutual funds that have either Pfizer or Moderna holdings: American Funds Fundamental, Federated Hermes Kaufman Fund, and Fidelity Biotechnology Fund. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Hosseinian, Powers, Vasudev, Palma, de Assis, Jain, Horvath, Birring, Andary, Au, Chin, Khalil, Ventura, Luu, Figueroa, Obiero, Silzel, Nakajima, Gombrich, Jasinskas, Zaldivar, Schubl, Felgner, Khan and The Specimen Collection Group.)

Background: COVID-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 Methods: This 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., Results: A/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., Conclusion: The 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., (© 2022 The Authors. Transfusion published by Wiley Periodicals LLC on behalf of AABB.)

Enzyme-linked immunosorbent assays (ELISAs) remain the gold standard for measuring antibodies, but are time-consuming and use significant amounts of precious sample and reagents. Protein microarrays represent an appealing alternative, particularly for studies focused on large gene families such as those encoding variant surface antigens in the malaria parasite Plasmodium falciparum. Such microarrays represent an ideal high-throughput platform to study antibody responses to hundreds of malaria parasite variant surface antigens at once, providing critical insights into the development of natural immunity to malaria. We describe the essential background and approach to run an assay using a P. falciparum microarray populated with variant surface antigens. This allows the user to define serologic profiles and identify serodominant antigens that represent promising targets for vaccine or therapeutic development., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Vaccination has been playing an important role in treating both infectious and cancerous diseases. Nevertheless, many diseases still lack proper vaccines due to the difficulty to generate sufficient amounts of antigen-specific antibodies or T cells. Adjuvants provide an important route to improve and direct immune responses. However, there are few adjuvants approved clinically and many of them lack the clear structure/adjuvanticity relationship. Here, we synthesized and evaluated a series of dendronized polypeptides (denpols) functionalized with varying tryptophan/histidine (W/H) molar ratios of 0/100, 25/75, 50/50, 75/25, and 100/0 as tunable synthetic adjuvants. The denpols showed structure-dependent inflammasome activation in THP1 monocytic cells and structure-related activation and antigen cross-presentation in vitro in bone marrow-derived dendritic cells. We used the denpols with bacterial pathogen Coxiella burnetii antigens in vivo , which showed both high and tunable adjuvating activities, as demonstrated by the antigen-specific antibody and T cell responses. The denpols are easy to make and scalable, biodegradable, and have highly adjustable chemical structures. Taken together, denpols show great potential as a new and versatile adjuvant platform that allows us to adjust adjuvanticity based on structure-activity correlation with the aim to fine-tune the immune response, thus advancing vaccine development.

We analyzed data from two ongoing COVID-19 longitudinal serological surveys in Orange County, CA., between April 2020 and March 2021. A total of 8476 finger stick blood specimens were collected before and after a vaccination campaign. IgG levels were determined using a multiplex antigen microarray containing antigens from SARS-CoV-2, SARS, MERS, Common CoV, and Influenza. Twenty-six percent of specimens from unvaccinated Orange County residents in December 2020 were SARS-CoV-2 seropositive; out of 852 seropositive individuals 77 had symptoms and 9 sought medical care. The antibody response was predominantly against nucleocapsid (NP), full length, and S2 domain of spike. Anti-receptor binding domain (RBD) reactivity was low and not cross-reactive against SARS S1 or SARS RBD. A vaccination campaign at the University of California Irvine Medical Center (UCIMC) started on December, 2020 and 6724 healthcare workers were vaccinated within 3 weeks. Seroprevalence increased from 13% pre-vaccination to 79% post-vaccination in January, 93% in February, and 99% in March. mRNA vaccination induced higher antibody levels than natural exposure, especially against the RBD domain and cross-reactivity against SARS RBD and S1 was observed. Nucleocapsid protein antibodies can be used to distinguish vaccinees to classify pre-exposure to SARS-CoV-2 Previously infected individuals developed higher antibody titers to the vaccine than non pre-exposed individuals. Hospitalized patients in intensive care with severe disease reach significantly higher antibody levels than mild cases, but lower antibody levels compared to the vaccine. These results indicate that mRNA vaccination rapidly induces a much stronger and broader antibody response than SARS-CoV-2 infection., (© 2021. The Author(s).)

Background: Sensitive diagnostics are needed for effective management and surveillance of schistosomiasis so that current transmission interruption goals set by WHO can be achieved. We aimed to screen the Schistosoma haematobium secretome to find antibody biomarkers of schistosome infection, validate their diagnostic performance in samples from endemic populations, and evaluate their utility as point of care immunochromatographic tests (POC-ICTs) to diagnose urogenital schistosomiasis in the field., Methods: We did a biomarker identification study, in which we constructed a proteome array containing 992 validated and predicted proteins from S haematobium and screened it with serum and urine antibodies from endemic populations in Gabon, Tanzania, and Zimbabwe. Arrayed antigens that were IgG-reactive and a select group of antigens from the worm extracellular vesicle proteome, predicted to be diagnostically informative, were then evaluated by ELISA using the same samples used to probe arrays, and samples from individuals residing in a low-endemicity setting (ie, Pemba and Unguja islands, Zanzibar, Tanzania). The two most sensitive and specific antigens were incorporated into POC-ICTs to assess their ability to diagnose S haematobium infection from serum in a field-deployable format., Findings: From array probing, in individuals who were infected, 208 antigens were the targets of significantly elevated IgG responses in serum and 45 antigens were the targets of significantly elevated IgG responses in urine. Of the five proteins that were validated by ELISA, Sh -TSP-2 (area under the curve [AUC] serum =0·98 [95% CI 0·95-1·00]; AUC urine =0·96 [0·93-0·99]), and MS3_01370 (AUC serum =0·93 [0·89-0·97]; AUC urine =0·81 [0·72-0·89]) displayed the highest overall diagnostic performance in each biofluid and exceeded that of S haematobium -soluble egg antigen in urine (AUC=0·79 [0·69-0·90]). When incorporated into separate POC-ICTs, Sh -TSP-2 showed absolute specificity and a sensitivity of 75% and MS3_01370 showed absolute specificity and a sensitivity of 89%., Interpretation: We identified numerous biomarkers of urogenital schistosomiasis that could form the basis of novel antibody diagnostics for this disease. Two of these antigens, Sh -TSP-2 and MS3_01370, could be used as sensitive, specific, and field-deployable diagnostics to support schistosomiasis control and elimination initiatives, with particular focus on post-elimination surveillance., Funding: Australian Trade and Investment Commission and Merck Global Health Institute., Competing Interests: MSP, AL, and JS are inventors on a provisional patent filed by the funding body that captures the data presented in this study. All other authors declare no competing interests., (© 2021 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY-NC-ND 4.0 license.)

We conducted a detailed analysis of coronavirus disease in a large population center in southern California, USA (Orange County, population 3.2 million), to determine heterogeneity in risks for infection, test positivity, and death. We used a combination of datasets, including a population-representative seroprevalence survey, to assess the actual burden of disease and testing intensity, test positivity, and mortality. In the first month of the local epidemic (March 2020), case incidence clustered in high-income areas. This pattern quickly shifted, and cases next clustered in much higher rates in the north-central area of the county, which has a lower socioeconomic status. Beginning in April 2020, a concentration of reported cases, test positivity, testing intensity, and seropositivity in a north-central area persisted. At the individual level, several factors (e.g., age, race or ethnicity, and ZIP codes with low educational attainment) strongly affected risk for seropositivity and death.

Background: Screening malaria-specific antibody responses on protein microarrays can help identify immune factors that mediate protection against malaria infection, disease, and transmission, as well as markers of past exposure to both malaria parasites and mosquito vectors. Most malaria protein microarray work has used serum as the sample matrix, requiring prompt laboratory processing and a continuous cold chain, thus limiting applications in remote locations. Dried blood spots (DBS) pose minimal biohazard, do not require immediate laboratory processing, and are stable at room temperature for transport, making them potentially superior alternatives to serum. The goals of this study were to assess the viability of DBS as a source for antibody profiling and to use DBS to identify serological signatures of low-density Plasmodium falciparum infections in malaria-endemic regions of Myanmar., Methods: Matched DBS and serum samples from a cross-sectional study in Ingapu Township, Myanmar were probed on protein microarrays populated with P. falciparum antigen fragments. Signal and trends in both sample matrices were compared. A case-control study was then performed using banked DBS samples from malaria-endemic regions of Myanmar, and a regularized logistic regression model was used to identify antibody signatures of ultrasensitive PCR-positive P. falciparum infections., Results: Approximately 30% of serum IgG activity was recovered from DBS. Despite this loss of antibody activity, antigen and population trends were well-matched between the two sample matrices. Responses to 18 protein fragments were associated with the odds of asymptomatic P. falciparum infection, albeit with modest diagnostic characteristics (sensitivity 58%, specificity 85%, negative predictive value 88%, and positive predictive value 52%)., Conclusions: Malaria-specific antibody responses can be reliably detected, quantified, and analysed from DBS, opening the door to serological studies in populations where serum collection, transport, and storage would otherwise be impossible. While test characteristics of antibody signatures were insufficient for individual diagnosis, serological testing may be useful for identifying exposure to asymptomatic, low-density malaria infections, particularly if sero-surveillance strategies target individuals with low previous exposure as sentinels for population exposure., (© 2021. The Author(s).)

Knowledge of the Plasmodium falciparum antigens that comprise the human liver stage immunoproteome is important for pre-erythrocytic vaccine development, but, compared with the erythrocytic stage immunoproteome, more challenging to classify. Previous studies of P. falciparum antibody responses report IgG and rarely IgA responses. We assessed IgG and IgA antibody responses in adult sera collected during two controlled human malaria infection (CHMI) studies in malaria-naïve volunteers and in 1- to 6-year-old malaria-exposed Malian children on a 251 P. falciparum antigen protein microarray. IgG profiles in the two CHMI groups were equivalent and differed from Malian children. IgA profiles were robust in the CHMI groups and a subset of Malian children. We describe immunoproteome differences in naïve vs. exposed individuals and report pre-erythrocytic proteins recognized by the immune system. IgA responses detected in this study expand the list of pre-erythrocytic antigens for further characterization as potential vaccine candidates., (© 2021. The Author(s).)

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., Competing Interests: JH-D, JF, EP, AaJ, SJ, RN, AlJ, PF, and DHD own shares in Nanommune Inc. Nanommune does not sell any products described in this paper, nor funded any part of the work described herein. Neither Nanommune nor its shareholders are likely to benefit from the results described in this publication. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Hernandez-Davies, Felgner, Strohmeier, Pone, Jain, Jan, Nakajima, Jasinskas, Strahsburger, Krammer, Felgner and Davies.)

Circumsporozoite protein (CSP) coats the Plasmodium falciparum sporozoite surface and is a major malaria subunit vaccine target. We measured epitope-specific reactivity to field-derived CSP haplotypes in serum samples from Malian adults and children on a custom peptide microarray. Compared to children, adults showed greater antibody responses and responses to more variants in regions proximal to and within the central repeat region. Children acquired short-lived immunity to an epitope proximal to the central repeat region but not to the central repeat region itself. This approach has the potential to differentiate immunodominant from protective epitope-specific responses when combined with longitudinal infection data., (© The Author(s) 2020. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

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 <0.0001) and intestinal egg burdens (50-54%, P <0.0003) were achieved in mice vaccinated with cystatin in two independent trials. This study has revealed numerous antigens that are targets of DIV antibodies in urogenital schistosomiasis and offer promise as subunit vaccine targets for a drug-linked vaccination approach to controlling schistosomiasis., Competing Interests: BG was employed by Ares Trading, S.A. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Pearson, Tedla, Becker, Nakajima, Jasinskas, Mduluza, Mutapi, Oeuvray, Greco, Sotillo, Felgner and Loukas.)

Plasmodium species, the causative agent of malaria, have a complex life cycle involving two hosts. The sporozoite life stage is characterized by an extended phase in the mosquito salivary glands followed by free movement and rapid invasion of hepatocytes in the human host. This transmission stage has been the subject of many transcriptomics and proteomics studies and is also targeted by the most advanced malaria vaccine. We applied Bayesian data integration to determine which proteins are not only present in sporozoites but are also specific to that stage. Transcriptomic and proteomic Plasmodium data sets from 26 studies were weighted for how representative they are for sporozoites, based on a carefully assembled gold standard for Plasmodium falciparum (Pf) proteins known to be present or absent during the sporozoite life stage. Of 5418 Pf genes for which expression data were available at the RNA level or at the protein level, 975 were identified as enriched in sporozoites and 90 specific to them. We show that Pf sporozoites are enriched for proteins involved in type II fatty acid synthesis in the apicoplast and GPI anchor synthesis, but otherwise appear metabolically relatively inactive in the salivary glands of mosquitos. Newly annotated hypothetical sporozoite-specific and sporozoite-enriched proteins highlight sporozoite-specific functions. They include PF3D7&#95;0104100 that we identified to be homologous to the prominin family, which in human has been related to a quiescent state of cancer cells. We document high levels of genetic variability for sporozoite proteins, specifically for sporozoite-specific proteins that elicit antibodies in the human host. Nevertheless, we can identify nine relatively well-conserved sporozoite proteins that elicit antibodies and that together can serve as markers for previous exposure. Our understanding of sporozoite biology benefits from identifying key pathways that are enriched during this life stage. This work can guide studies of molecular mechanisms underlying sporozoite biology and potential well-conserved targets for marker and drug development., Competing Interests: The authors have declared that no competing interests exist.

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., (Copyright © 2021 Sen et al.)

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.

Q fever is caused by the obligate intracellular bacterium, Coxiella burnetii , a designated potential agent of bioterrorism because of its route of transmission, resistance to disinfectants, and low infectious dose. The only vaccine licensed for human use is Q-VAX ® (Seqirus, licensed in Australia), a formalin-inactivated whole-cell vaccine, which produces severe local and systemic reactogenic responses in previously sensitized individuals. Accordingly, the U.S. Food and Drug Administration and other regulatory bodies around the world, have been reluctant to approve Q-VAX for widespread use. To obviate these adverse reactions, we prepared recombinant protein subunit vaccine candidates containing purified CBU1910, CBU0307, CBU0545, CBU0612, CBU0891, and CBU1398 proteins and TLR triagonist adjuvants. TLR triagonist adjuvants combine different TLR agonists to enhance immune responses to vaccine antigens. We tested both the protective efficacy and reactogenicity of our vaccine candidates in Hartley guinea pigs using intratracheal infection with live C. burnetii . While all of our candidates showed varying degrees of protection during challenge, local reactogenic responses were significantly reduced for one of our vaccine candidates when compared with a formalin-inactivated whole-cell vaccine. Our findings show that subunit vaccines combined with novel TLR triagonist adjuvants can generate protective immunity to C. burnetii infection while reducing reactogenic responses., Competing Interests: PF, DD, JF, LL, SJ, RN, and AJ own shares in Nanommune Inc. Nanommune does not sell the arrays described in this paper, nor funded any part of the work described herein. Neither Nanommune or its shareholders are likely to benefit from the results described in this publication. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Fratzke, Jan, Felgner, Liang, Nakajima, Jasinskas, Manna, Nihesh, Maiti, Albin, Esser-Kahn, Davies, Samuel, Felgner and Gregory.)

Leptospirosis is the leading zoonotic disease in terms of morbidity and mortality worldwide. Effective prevention is urgently needed as the drivers of disease transmission continue to intensify. The key challenge has been developing a widely applicable vaccine that protects against the >300 serovars that can cause leptospirosis. Live attenuated mutants are enticing vaccine candidates and poorly explored in the field. We evaluated a recently characterized motility-deficient mutant lacking the expression of a flagellar protein, FcpA. Although the fcpA - mutant has lost its ability to cause disease, transient bacteremia was observed. In two animal models, immunization with a single dose of the fcpA - mutant was sufficient to induce a robust anti-protein antibodies response that promoted protection against infection with different pathogenic Leptospira species. Furthermore, characterization of the immune response identified a small repertoire of biologically relevant proteins that are highly conserved among pathogenic Leptospira species and potential correlates of cross-protective immunity., Competing Interests: EW, HA, CH, KO, LL, CR, VB, JN, DA, MR, PD, PF, AK No competing interests declared

Background: Shigella is a major diarrheal pathogen for which there is presently no vaccine. Whole genome sequencing provides the ability to predict and derive novel antigens for use as vaccines. Here, we aimed to identify novel immunogenic Shigella antigens that could serve as Shigella vaccine candidates, either alone, or when conjugated to Shigella O-antigen., Methods: Using a reverse vaccinology approach, where genomic analysis informed the Shigella immunome via an antigen microarray, we aimed to identify novel immunogenic Shigella antigens. A core genome analysis of Shigella species, pathogenic and non-pathogenic Escherichia coli, led to the selection of 234 predicted immunogenic Shigella antigens. These antigens were expressed and probed with acute and convalescent serum from microbiologically confirmed Shigella infections., Results: Several Shigella antigens displayed IgG and IgA seroconversion, with no difference in sero-reactivity across by sex or age. IgG sero-reactivity to key Shigella antigens was observed at birth, indicating transplacental antibody transfer. Six antigens (FepA, EmrK, FhuA, MdtA, NlpB, and CjrA) were identified in in vivo testing as capable of producing binding IgG and complement-mediated bactericidal antibody., Conclusions: These findings provide six novel immunogenic Shigella proteins that could serve as candidate vaccine antigens, species-specific carrier proteins, or targeted adjuvants.

The current practice for diagnosis of COVID-19, based on SARS-CoV-2 PCR testing of pharyngeal or respiratory specimens in a symptomatic patient at high epidemiologic risk, likely underestimates the true prevalence of infection. Serologic methods can more accurately estimate the disease burden by detecting infections missed by the limited testing performed to date. Here, we describe the validation of a coronavirus antigen microarray containing immunologically significant antigens from SARS-CoV-2, in addition to SARS-CoV, MERS-CoV, common human coronavirus strains, and other common respiratory viruses. A comparison of antibody profiles detected on the array from control sera collected prior to the SARS-CoV-2 pandemic versus convalescent blood specimens from virologically confirmed COVID-19 cases demonstrates near complete discrimination of these two groups, with improved performance from use of antigen combinations that include both spike protein and nucleoprotein. This array can be used as a diagnostic tool, as an epidemiologic tool to more accurately estimate the disease burden of COVID-19, and as a research tool to correlate antibody responses with clinical outcomes.

The protein array is a powerful platform to study humoral responses to infectious agents using small sample volumes [<3 μL]. Its success can be largely attributed to the development of new strategies for high-throughput cloning and expression, and improved manufacturing techniques for the construction of arrays. Here, we describe a method to hybridize protein arrays with malaria patients' sera in order to identify seroreactive antigens, some of which may have a high potential of conferring protection from severe forms of malaria.

Most of what we know about adaptive immunity has come from inbred mouse studies, using methods that are often difficult or impossible to confirm in humans. In addition, vaccine responses in mice are often poorly predictive of responses to those same vaccines in humans. Here we use human tonsils, readily available lymphoid organs, to develop a functional organotypic system that recapitulates key germinal center features in vitro, including the production of antigen-specific antibodies, somatic hypermutation and affinity maturation, plasmablast differentiation and class-switch recombination. We use this system to define the essential cellular components necessary to produce an influenza vaccine response. We also show that it can be used to evaluate humoral immune responses to two priming antigens, rabies vaccine and an adenovirus-based severe acute respiratory syndrome coronavirus 2 vaccine, and to assess the effects of different adjuvants. This system should prove useful for studying critical mechanisms underlying adaptive immunity in much greater depth than previously possible and to rapidly test vaccine candidates and adjuvants in an entirely human system.

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., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

To detect the presence of antibodies in blood against SARS-CoV-2 in a highly sensitive and specific manner, here we describe a robust, inexpensive ($200), 3D-printable portable imaging platform (TinyArray imager) that can be deployed immediately in areas with minimal infrastructure to read coronavirus antigen microarrays (CoVAMs) that contain a panel of antigens from SARS-CoV-2, SARS-1, MERS, and other respiratory viruses. Application includes basic laboratories and makeshift field clinics where a few drops of blood from a finger prick could be rapidly tested in parallel for the presence of antibodies to SARS-CoV-2 with a test turnaround time of only 2-4 h. To evaluate our imaging device, we probed and imaged coronavirus microarrays with COVID-19-positive and negative sera and achieved a performance on par with a commercial microarray reader 100× more expensive than our imaging device. This work will enable large scale serosurveillance, which can play an important role in the months and years to come to implement efficient containment and mitigation measures, as well as help develop therapeutics and vaccines to treat and prevent the spread of COVID-19.

Soluble oligomers of aggregated tau accompany the accumulation of insoluble amyloid fibrils, a histological hallmark of Alzheimer disease (AD) and two dozen related neurodegenerative diseases. Both oligomers and fibrils seed the spread of Tau pathology, and by virtue of their low molecular weight and relative solubility, oligomers may be particularly pernicious seeds. Here, we report the formation of in vitro tau oligomers formed by an ionic liquid (IL15). Using IL15-induced recombinant tau oligomers and a dot blot assay, we discovered a mAb (M204) that binds oligomeric tau, but not tau monomers or fibrils. M204 and an engineered single-chain variable fragment (scFv) inhibited seeding by IL15-induced tau oligomers and pathological extracts from donors with AD and chronic traumatic encephalopathy. This finding suggests that M204-scFv targets pathological structures that are formed by tau in neurodegenerative diseases. We found that M204-scFv itself partitions into oligomeric forms that inhibit seeding differently, and crystal structures of the M204-scFv monomer, dimer, and trimer revealed conformational differences that explain differences among these forms in binding and inhibition. The efficiency of M204-scFv antibodies to inhibit the seeding by brain tissue extracts from different donors with tauopathies varied among individuals, indicating the possible existence of distinct amyloid polymorphs. We propose that by binding to oligomers, which are hypothesized to be the earliest seeding-competent species, M204-scFv may have potential as an early-stage diagnostic for AD and tauopathies, and also could guide the development of promising therapeutic antibodies., Competing Interests: Conflict of interest—D. S. E. is SAB chair and an equity holder of ADRx, Inc., (© 2020 Abskharon et al.)

Here, we describe a serological enzyme-linked immunosorbent assay for the screening and identification of human SARS-CoV-2 seroconverters. This assay does not require the handling of infectious virus, can be adjusted to detect different antibody types in serum and plasma and is amenable to scaling. Serological assays are of critical importance to help define previous exposure to SARS-CoV-2 in populations, identify highly reactive human donors for convalescent plasma therapy and investigate correlates of protection.

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.