BACKGROUND:The 2022 global outbreak of monkeypox virus (MPXV) clade IIb prompted widespread use of the modified vaccinia Ankara-Bavarian Nordic (MVA-BN) vaccine which conferred partial protection and contributed to outbreak control. In 2024, the emergence and regional expansion of MPXV clade Ib in central Africa raised new public health concerns and prompted questions about the extent to which immunity elicited by previous clade IIb infection or vaccination confers cross-neutralising immunity against clade Ib. We aimed to assess neutralising and binding antibody responses to MPXV clades Ia, Ib, and IIb after infection or vaccination. METHODS:In this multicountry, observational study, plasma samples were collected from three academic medical sites in the USA, Brazil, and Portugal between July 7, 2022, and Aug 3, 2023. Individuals contributing samples were categorised into five cohorts based on their vaccination and infection status: individuals vaccinated with a single dose of the first-generation smallpox vaccine Dryvax, individuals vaccinated with MVA-BN, individuals vaccinated with Dryvax and MVA-BN, individuals with PCR-confirmed MPXV clade IIb infection, and individuals who were neither vaccinated nor had a previous MPXV infection (control cohort). For individuals vaccinated with Dryvax, vaccination occurred during childhood between 1961 and 1987. For individuals in the MVA-BN and MVA-BN plus Dryvax cohorts, MVA-BN was administered at participating sites between July 7, 2022, and Aug 3, 2023. Plasma samples were collected at a single timepoint in the Dryvax, MPXV clade IIb infection, and control cohorts and at multiple predefined timepoints in the MVA-BN and MVA-BN plus Dryvax cohorts. Individuals younger than 18 years and pregnant people were excluded. Individuals in the vaccination cohorts had no history of MPXV infection or exposure. Antibody binding was measured by ELISA with inactivated whole-virus antigens and recombinant proteins; neutralisation activity was assessed by plaque reduction neutralisation tests (PRNT50). Vaccina virus (VACV) was included as a reference control for vaccine-induced immunity. For ELISA and neutralisation assays, thresholds of positivity were defined as the median plus 3 SDs of values obtained from individuals in the control cohort. FINDINGS:All eligible participants with available plasma samples meeting inclusion criteria were included in the study. We analysed 232 plasma samples from 191 participants: 40 samples from 40 individuals in the Dryvax cohort, 62 samples from 33 individuals in the MVA-BN cohort, 31 samples from 19 individuals in the Dryvax plus MVA-BN cohort, 77 samples from 77 individuals in the MPXV clade IIb cohort, and 22 samples from 22 individuals in the control cohort. Across vaccinated cohorts, polyclonal plasma antibodies exhibited cross-reactive binding to MPXV clades Ia, Ib, and IIb, with detectable binding observed in 33-81% of plasma samples, depending on vaccine regimen. Detectable MPXV neutralisation (PRNT50 above threshold) was observed in 28-35% of plasma samples from the Dryvax cohort and 39-55% of plasma samples from the Dryvax plus MVA-BN cohort, whereas responder frequencies were lower among plasma samples from the MVA-BN cohort (3-15%). Median PRNT50 values against MPXV clades Ia, Ib, and IIb ranged from 1·00 to 1·03 across vaccination cohorts, whereas higher neutralisation values were observed against VACV (ranging from 1·48 to 2·22 across cohorts). Individuals in the MPXV clade IIb infection cohort exhibited broad antibody binding but low neutralisation of MPXV clade Ib virus (median log10PRNT50 1·02 [1·02-1·30] for clade Ia, 1·00 [1·00-1·00] for clade Ib, and 1·20 [IQR 1·00-1·76] for clade IIb). At the antigen level, A35-targeted antibodies bound less efficiently to clade I A35 variants compared with clade IIb. INTERPRETATION:Humoral immunity elicited by historical or contemporary smallpox vaccination was associated with little neutralising activity across MPXV clades. Of note, previous MPXV clade IIb infection induced cross-reactive antibody binding but low-magnitude neutralising activity against clade Ib. Although additional immune mechanisms could contribute to protection, these findings highlight important constraints in antibody-mediated cross-clade immunity and emphasise the need for vaccines and antibody-based countermeasures that address antigenic divergence among orthopoxviruses. FUNDING:Yale University, Stavros Niarchos Foundation Institute for Global Infectious Disease, Icahn School of Medicine at Mount Sinai, US National Institutes of Health, and US National Institute of Allergy and Infectious Diseases.

The SARS-CoV-2 pandemic underscored the need for innovative approaches to study humoral immunity and isolate monoclonal antibodies (mAbs) with diagnostic and therapeutic potential. Current methods for repertoire analysis at the clonal level require large-scale recombinant mAb production, limiting accessibility and delaying functional insight. We developed a single-cell culture (SCC) platform that enables profiling of human memory B cells and direct recovery of functional mAbs. Using samples from COVID-19 convalescent and vaccinated donors, we optimized SCCs with NB21 feeder cells, R848, and IL-2, achieving efficient clonal expansion and antibody secretion in short-term cultures. Screening and pseudovirus neutralization assays were performed directly with culture supernatants, bypassing the need for early recombinant antibody production. Antigen-baited cytometry sorting enriched spike-specific memory B cells by ∼30-fold. Among 592 isolated mAbs, 53% bound the Wuhan spike, targeting the receptor-binding domain (28%), N-terminal domain (15%), or other regions (57%). Cross-reactivity analysis revealed that 40% of anti-spike mAbs recognized all tested variants of concern. VH/VL sequencing uncovered convergent rearrangements, including public V3-30 and V3-53/V3-66 clones, consistent with global findings. Two public receptor-binding domain-specific antibodies demonstrated broad neutralization when produced recombinantly. Together, these results validate the SCC system as a streamlined approach for unbiased repertoire analysis and functional mAb isolation. More broadly, the platform provides a practical framework for linking B cell clonal composition with antigen specificity and serum antibody responses. By reducing costs and simplifying workflows, it expands opportunities for antibody discovery and immunoepidemiological studies, fostering wider global participation in therapeutic antibody research.

Background:After decades of circulating predominantly in Africa, mpox caused a public health emergency of international concern between 2022 and 2023. Even outside of the epidemic period, the virus still circulates, with threats of reemergence, and different clinical presentations must be brought to attention for optimal surveillance. Case Summary:We report a case of monkeypox virus (MPXV) infection in a previously healthy physician with an atypical clinical presentation and a single identified exposure. Diagnosis was confirmed by PCR. Viral isolation was employed to reinforce diagnosis. Plaque reduction neutralization assays and semiquantitative ELISA were used to evaluate the antibody response. Conclusion:Our report has two main messages: atypical symptoms of mpox can hinder diagnosis, making adequate suspicion and testing fundamental; and the risk of occupational exposure must be considered when designing policies for healthcare workers.

BACKGROUND:Cross-reactive immune memory responses to orthopoxviruses in humans remain poorly characterised despite their relevance for vaccine design and outbreak control. We aimed to assess the magnitude, specificity, and durability of cross-reactive immune responses elicited by smallpox vaccines and mpox virus infection. METHODS:We did a multicohort observational study involving participants from the USA, Brazil, and Portugal across four groups: Dryvax (first-generation smallpox vaccine) recipients vaccinated 40-80 years ago, JYNNEOS (third-generation smallpox vaccine) recipients vaccinated within the past year, a cohort receiving both vaccines, and patients infected with clade IIb mpox. Samples were analysed for systemic and mucosal humoral responses, neutralising antibody titres, viral antigen structural analysis, and T-cell cross-reactivity to vaccina virus, cowpox virus, and mpox virus. Statistical analyses included correlation assessments and comparisons across cohorts to determine the magnitude, longevity, and breadth of immune responses. FINDINGS:Between July 7, 2022, and Aug 3, 2023, 262 participants were recruited, resulting in analysis of 378 samples. Both first-generation and third-generation smallpox vaccines elicited vaccinia virus-reactive and mpox virus-reactive antibodies, with the strongest responses targeting the less conserved extracellular virion antigens B5 and A33. Despite high concentrations of anti-mpox virus antibodies in the plasma, cross-neutralisation activity correlated with viral antigenic distance. Higher neutralisation was observed for cowpox virus than for mpox virus, which has lower antigenic conservation with vaccina virus. Complement-mediated neutralisation enhanced mpox virus neutralisation, overcoming the limitations of antigenic distance. Dryvax recipients sustained vaccina virus neutralisation titres for over 80 years, whereas cross-reactive responses did not show this durability. JYNNEOS-induced responses waned within a year. T-cell cross-reactivity was long-lasting, detected up to 70 years after vaccination. Booster vaccinations augmented the magnitude, breadth, and longevity of cross-neutralising responses. INTERPRETATION:Our findings highlight the potential combined role of antibody effector functions and T-cell memory in cross-protection against orthopoxviruses. Complement-mediated neutralisation enhances cross-protection, overcoming antigenic distance. These Fc-mediated functions, along with T-cell responses, contribute to effective and long-lasting immunity conferred by smallpox vaccines against other orthopoxviruses. FUNDING:Yale University and Stavros Niarchos Foundation Institute for Global Infectious Disease.

The SARS-CoV-2 pandemic highlighted the urgent need for innovative methods to study humoral immune responses and identify monoclonal antibodies (mAbs) with diagnostic and therapeutic potential. Memory B cells (MBCs), pivotal to adaptive immunity, generate high-affinity antibodies upon antigen re-encounter. While single-cell high-throughput sequencing has revolutionized antibody repertoire studies, it has critical limitations: the inability to simultaneously determine antigen-binding specificities and immunoglobulin gene sequences, and high resource demands that limit accessibility in low-resource settings. Here, we present a cost-effective single-cell culture (SCC) platform enabling comprehensive analysis of human MBC repertoires, including epitope-specific responses, cross-reactivity studies, and mAb isolation. Using SARS-CoV-2 convalescent and vaccinated donor samples, we optimized MBC SCCs with NB21 feeder cells, R848, and IL-2 stimulation, achieving high cloning efficiency and a 30-fold enrichment of antigen-specific MBCs compared to bulk cultures. Among 592 isolated mAbs, 52.7% exhibited specificity to the Wuhan strain Spike protein, with 27.9% targeting the receptor-binding domain (RBD), 15.4% the N-terminal domain (NTD), and 56.7% other regions, likely the S2 domain. Comparative analysis revealed distinct cross-reactivity patterns: 40.5% of all anti-Spike mAbs recognized all tested SARS-CoV-2 variants (Wuhan, Beta, Delta, Gamma and Omicron BA.2), while 29.6% showed recognition of only four variants, the majority not including Omicron BA.2; 56 single strain reactive mAbs (14.9%) were also identified. Notably, all screening and neutralization assays were performed directly with culture supernatants, eliminating the need for large-scale sequencing and transfection. Desired clones were selected for recombinant mAb production. The SCC platform also enabled unbiased immunoglobulin repertoire profiling, revealing convergent V-region rearrangements, including public V3-30 and V3-53/V3-66 antibodies consistent with prior SARS-CoV-2 studies. Two public RBD-targeting clones with broad neutralization potential were validated in pseudovirus neutralization assays. This streamlined platform delivers simultaneous antigen-specific mAb isolation, V-region sequencing, and functional studies within seven days, empowering researchers in low-resource settings to address global health inequities and enhance preparedness for future pandemics. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement GIISER-Brazil/BMGF partially funded this work to ACB and AMV; the Stavros Niarchos Foundation Institute at Rockefeller University (SNIFRU) to GDV and AMV; and the Brazilian research funding agencies: Fundacao de Amparo a Pesquisa do Estado do Rio de Janeiro (FAPERJ), Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq), Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES), Financiadora de Estudos e Projetos (FINEP) and Fundacao de Amparo a Pesquisa de Minas Gerais (FAPEMIG) [APQ-00501-23, APQ-04025-23, Rede Mineira de Imunobiologicos grant REDE-00140-16]. L. Conde received a PhD fellowship from FAPERJ. Additional funding was provided by INOVA COVID-19/FIOCRUZ, INCT NeuroImmunomodulation, Rede de ImunoInflamacao, and FOCEM/Mercosul to ACB; Instituto Serrapilheira grant to AT; CAPES (COMBATECovid) and ANRS | Maladies infectieuses emergentes/Inserm grant (MUCOVID- 007) grant to MTB; Corona-omica -CNPq/FINEP to LJC. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: This study was approved by the National Ethics Committee (CONEP, Brazil; CAAE:30161620.0.0000.5257) and the local ethics committee of Fundacao Oswaldo Cruz (CAAE:3048.7120.2.0000.0008). I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors

Immunological memory mediates rapid protection following infection or vaccination including heterologous exposure. However, cross-reactive memory responses in humans remain poorly characterized. We explored the longevity and specificity of cross-protective responses to orthopoxviruses through smallpox vaccination and Mpox virus (MPXV) infection. Smallpox vaccination using Vaccinia virus (VACV)-based vaccines provides a unique opportunity to study long-term cross-protective immunity without antigen re-exposure. We assessed systemic and mucosal responses in four human cohorts, including first-(Dryvax) and/or third-generation (JYNNEOS) smallpox vaccine recipients (vaccinated 1 week-80 years ago), along with Mpox-infected individuals. First-and third-generation smallpox vaccines elicited strong VACV- and MPXV-specific antibodies. VACV-neutralizing antibodies persisted for decades in first-generation vaccine recipients and were further enhanced after JYNNEOS vaccination. However, despite the high levels of anti-MPXV-specific antibodies in the plasma, cross-neutralization activity was directly correlated with the antigenic distance. Higher neutralization was observed for the cowpox virus (CWPXV) than for MPXV, which showed lower antigenic conservation with VACV. Similarly, Mpox-infected patients had lower neutralization titers for VACV than for CWPXV. Individuals who received vaccination boosters showed more robust, diverse, and prolonged cross-neutralizing responses. Long-term memory analysis revealed an increase in neutralization capacity for VACV over decades, with 80-years-old displaying the most robust humoral response, although this trend was not observed for cross-reactive antigens. Finally, T-cell reactivity to VACV and MPXV epitopes was detected decades post-vaccination, suggesting a role of long-lasting cross-reactive T-cell memory responses in vaccine efficacy. Our findings underscore the pivotal influence of antigenic distance on vaccine effectiveness with implications for cross-protective vaccine design.

The vast spectrum of clinical features of COVID-19 keeps challenging scientists and clinicians. Low resistance to infection might result in long-term viral persistence, but the underlying mechanisms remain unclear. Here, we studied the immune response of immunocompetent COVID-19 patients with prolonged SARS-CoV-2 infection by immunophenotyping, cytokine and serological analysis. Despite viral loads and symptoms comparable to regular mildly symptomatic patients, long-term carriers displayed weaker systemic IFN-I responses and fewer circulating pDCs and NK cells at disease onset. Type 1 cytokines remained low, while type-3 cytokines were in turn enhanced. Of interest, we observed no defects in antigen-specific cytotoxic T cell responses, and circulating antibodies displayed higher affinity against different variants of SARS-CoV-2 Spike protein in these patients. The identification of distinct immune responses in long-term carriers adds up to our understanding of essential host protective mechanisms to ensure tissue damage control despite prolonged viral infection.

Although brain scars in adults have been extensively studied, there is less data available regarding scar formation during the neonatal period, and the involvement of peripheral immune cells in this process remains unexplored in neonates. Using a murine model of neonatal hypoxic-ischemic encephalopathy (HIE) and confocal microscopy, we characterized the scarring process and examined the recruitment of peripheral immune cells to cortical and hippocampal scars for up to 1 year post-insult. Regional differences in scar formation were observed, including the presence of reticular fibrotic networks in the cortex and perivascular fibrosis in the hippocampus. We identified chemokines with chronically elevated levels in both regions and demonstrated, through a parabiosis-based strategy, the recruitment of lymphocytes, neutrophils, and monocyte-derived macrophages to the scars several weeks after the neonatal insult. After 1 year, however, neutrophils and lymphocytes were absent from the scars. Our data indicate that peripheral immune cells are transient components of HIE-induced brain scars, opening up new possibilities for late therapeutic interventions.

Leishmaniasis presents different types of clinical manifestations that can be divided into cutaneous leishmaniasis and visceral leishmaniasis. The host’s immune system, associated with genetic and nutritional factors, is strongly involved in the evolution of the disease or parasite escape. Humoral immunity is characterized by the production of antibodies capable of promoting neutralization, opsonization, and activation of the complement system. In this scenario, B lymphocytes produce antibodies that play an important role in Leishmania infection although neglected for a long time. Thus, relevant aspects in the establishment of Leishmania infection will be addressed, highlighting the importance of humoral immunity during the entire process of Leishmania infection.

Graft-versus-host disease (GVHD) is the main complication of bone marrow transplantation (BMT). CD4+ T lymphocytes are the main effector cells for disease development, but other cell types can determine disease outcome through cytokine production and antigen presentation. B cells are abundant in BMT products and are involved in chronic GVHD immunopathogenesis. However, their role in acute GVHD is still unclear. Here we studied the role of donor resting B cells in a model of acute GVHD. Animals receiving transplants depleted of B cells developed more severe disease, indicating a protective role for B cells. Mice undergoing transplantation with IL-10 knockout B cells developed GVHD as severe as those receiving wild-type B cells. Moreover, mice that received MHC II-deficient B cells, and thus were unable to present antigen to CD4+ T cells, developed as severe GVHD as animals receiving transplants without B cells. This result suggests that the protection provided by mature naive B cells depends on antigen presentation and not on IL-10 production by B cells. Mice who underwent transplantation in the absence of donor B cells exhibited disorganized lymphoid splenic tissue. In addition, donor B cell depletion diminished the follicular T (Tfh)/effector T (Teff) cell ratio, suggesting that protection was correlated with a shift to Tfh differentiation, reducing the number of Teff cells. Importantly, the Tfh/Teff shift impacts disease outcome, with observed proinflammatory cytokine levels and tissue damage in target organs consistent with disease protection. The role of transplanted B cells in the outcome of BMT and the development of acute GVHD merits careful study, given that these cells are abundant in BMT products and are potent modulator and effector cells in the allogeneic response.

Despite the intramuscular route being the most used vaccination strategy against SARS-CoV-2, the intradermal route has been studied around the globe as a strong candidate for immunization against SARS-CoV-2. Adjuvants have shown to be essential vaccine components that are capable of driving robust immune responses and increasing the vaccination efficacy. In this work, our group aimed to develop a vaccination strategy for SARS-CoV-2 using a trimeric spike protein, by testing the best route with formulations containing the adjuvants AddaS03, CpG, MPL, Alum, or a combination of two of them. Our results showed that formulations that were made with AddaS03 or CpG alone or AddaS03 combined with CpG were able to induce high levels of IgG, IgG1, and IgG2a; high titers of neutralizing antibodies against SARS-CoV-2 original strain; and also induced high hypersensitivity during the challenge with Spike protein and a high level of IFN-γ producing CD4+ T-cells in mice. Altogether, those data indicate that AddaS03, CpG, or both combined may be used as adjuvants in vaccines for COVID-19.

The SARS-CoV-2 pandemic has had a social and economic impact worldwide, and vaccination is an efficient strategy for diminishing those damages. New adjuvant formulations are required for the high vaccine demands, especially adjuvant formulations that induce a Th1 phenotype. Herein we assess a vaccination strategy using a combination of Alum and polyinosinic:polycytidylic acid [Poly(I:C)] adjuvants plus the SARS-CoV-2 spike protein in a prefusion trimeric conformation by an intradermal (ID) route. We found high levels of IgG anti-spike antibodies in the serum by enzyme linked immunosorbent assay (ELISA) and high neutralizing titers against SARS-CoV-2 in vitro by neutralization assay, after two or three immunizations. By evaluating the production of IgG subtypes, as expected, we found that formulations containing Poly(I:C) induced IgG2a whereas Alum did not. The combination of these two adjuvants induced high levels of both IgG1 and IgG2a. In addition, cellular immune responses of CD4+ and CD8+ T cells producing interferon-gamma were equivalent, demonstrating that the Alum + Poly(I:C) combination supported a Th1 profile. Based on the high neutralizing titers, we evaluated B cells in the germinal centers, which are specific for receptor-binding domain (RBD) and spike, and observed that more positive B cells were induced upon the Alum + Poly(I:C) combination. Moreover, these B cells produced antibodies against both RBD and non-RBD sites. We also studied the impact of this vaccination preparation [spike protein with Alum + Poly(I:C)] in the lungs of mice challenged with inactivated SARS-CoV-2 virus. We found a production of IgG, but not IgA, and a reduction in neutrophil recruitment in the bronchoalveolar lavage fluid (BALF) of mice, suggesting that our immunization scheme reduced lung inflammation. Altogether, our data suggest that Alum and Poly(I:C) together is a possible adjuvant combination for vaccines against SARS-CoV-2 by the intradermal route.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to be a global problem in part because of the emergence of variants of concern that evade neutralization by antibodies elicited by prior infection or vaccination. Here we report on human neutralizing antibody and memory responses to the Gamma variant in a cohort of hospitalized individuals. Plasma from infected individuals potently neutralized viruses pseudotyped with Gamma SARS-CoV-2 spike protein, but neutralizing activity against Wuhan-Hu-1-1, Beta, Delta, or Omicron was significantly lower. Monoclonal antibodies from memory B cells also neutralized Gamma and Beta pseudoviruses more effectively than Wuhan-Hu-1. 69% and 34% of Gamma-neutralizing antibodies failed to neutralize Delta or Wuhan-Hu-1. Although Class 1 and 2 antibodies dominate the response to Wuhan-Hu-1 or Beta, 54% of antibodies elicited by Gamma infection recognized Class 3 epitopes. The results have implications for variant-specific vaccines and infections, suggesting that exposure to variants generally provides more limited protection to other variants.

Serological tests detect antibodies generated by infection or vaccination, and are indispensable tools along different phases of a pandemic, from early monitoring of pathogen spread up to seroepidemiological studies supporting immunization policies. This work discusses the development of an accurate and affordable COVID-19 antibody test, from production of a recombinant protein antigen up to test validation and economic analysis. We first developed a cost-effective, scalable technology to produce SARS-COV-2 spike protein and then used this antigen to develop an enzyme-linked immunosorbent assay (ELISA). A receiver operator characteristic (ROC) analysis allowed optimizing the cut-off and confirmed the high accuracy of the test: 98.6% specificity and 95% sensitivity for 11+ days after symptoms onset. We further showed that dried blood spots collected by finger pricking on simple test strips could replace conventional plasma/serum samples. A cost estimate was performed and revealed a final retail price in the range of one US dollar, reflecting the low cost of the ELISA test platform and the elimination of the need for venous blood sampling and refrigerated sample handling in clinical laboratories. The presented workflow can be completed in 4 months from first antigen expression to final test validation. It can be applied to other pathogens and in future pandemics, facilitating reliable and affordable seroepidemiological surveillance also in remote areas and in low-income countries.

Summary The vast spectrum of clinical features of COVID-19 keeps challenging scientists and clinicians. Control of pathogen load (host resistance) and prevention of tissue damage (disease tolerance) are essential for the outcome of infectious diseases. Both low resistance and high disease tolerance might result in long-term viral persistence, but the underlying mechanisms remain unclear. Here, we studied the immune response of immunocompetent COVID-19 patients with prolonged SARS-CoV-2 infection by immunophenotyping, cytokine and serological analysis. Despite viral loads and symptoms comparable to regular mildly-symptomatic patients, long-term carriers displayed weaker systemic IFN-I responses and fewer circulating pDCs and NK cells at disease onset. Type 1 cytokines remained low, while type-3 cytokines were in turn enhanced. Interestingly, the plasma of these patients showed a higher spike-specific neutralization capacity. The identification of very early distinct immune responses in long-term carriers adds up to our understanding on essential host protective mechanisms to ensure tissue damage control despite prolonged viral infection.

Besides antigen-specific responses to viral antigens, humoral immune response in virus infection can generate polyreactive and autoreactive antibodies. Dengue and Zika virus infections have been linked to antibody-mediated autoimmune disorders, including Guillain-Barré syndrome. A unique feature of flaviviruses is the secretion of nonstructural protein 1 (NS1) by infected cells. NS1 is highly immunogenic, and antibodies targeting NS1 can have both protective and pathogenic roles. In the present study, we investigated the humoral immune response to Zika virus NS1 and found NS1 to be an immunodominant viral antigen associated with the presence of autoreactive antibodies. Through single B cell cultures, we coupled binding assays and BCR sequencing, confirming the immunodominance of NS1. We demonstrate the presence of self-reactive clones in germinal centers after both infection and immunization, some of which present cross-reactivity with NS1. Sequence analysis of anti-NS1 B cell clones showed sequence features associated with pathogenic autoreactive antibodies. Our findings demonstrate NS1 immunodominance at the cellular level as well as a potential role for NS1 in ZIKV-associated autoimmune manifestations.

The dynamics underlying severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) reinfection remain poorly understood. We identified a small cluster of patients in Brazil who experienced 2 episodes of coronavirus disease (COVID-19) in March and late May 2020. In the first episode, patients manifested an enhanced innate response compared with healthy persons, but neutralizing humoral immunity was not fully achieved. The second episode was associated with different SARS-CoV-2 strains, higher viral loads, and clinical symptoms. Our finding that persons with mild COVID-19 may have controlled SARS-CoV-2 replication without developing detectable humoral immunity suggests that reinfection is more frequent than supposed, but this hypothesis is not well documented.

Background: Convalescent plasma is a potential therapeutic option for critically ill patients with coronavirus disease 19 (COVID-19), yet its efficacy remains to be determined. The aim was to investigate the effects of convalescent plasma (CP) in critically ill patients with COVID-19.Methods: This was a single-center prospective observational study conducted in Rio de Janeiro, Brazil, from March 17th to May 30th, with final follow-up on June 30th. We included 113 laboratory-confirmed COVID-19 patients with respiratory failure. Primary outcomes were time to clinical improvement and survival within 28 days. Secondary outcomes included behavior of biomarkers and viral loads. Kaplan–Meier analyses and Cox proportional-hazards regression using propensity score with inverse-probability weighing were performed.Results: 41 patients received CP and 72 received standard of care (SOC). Median age was 61 years (IQR 48–68), disease duration was 10 days (IQR 6–13), and 86% were mechanically ventilated. At least 29 out of 41CP-recipients had baseline IgG titers ≥ 1:1,080. Clinical improvement within 28 days occurred in 19 (46%) CP-treated patients, as compared to 23 (32%) in the SOC group [adjusted hazard ratio (aHR) 0.91 (0.49–1.69)]. There was no significant change in 28-day mortality (CP 49% vs. SOC 56%; aHR 0.90 [0.52–1.57]). Biomarker assessment revealed reduced inflammatory activity and increased lymphocyte count after CP.Conclusions: In this study, CP was not associated with clinical improvement or increase in 28-day survival. However, our study may have been underpowered and included patients with high IgG titers and life-threatening disease.Clinical Trial Registration: The study protocol was retrospectively registered at the Brazilian Registry of Clinical Trials (ReBEC) with the identification RBR-4vm3yy (http://www.ensaiosclinicos.gov.br).

Extracellular vesicles (EVs) are lipid bilayered compartments released by virtually all living cells, including fungi. Among the diverse molecules carried by fungal EVs, a number of immunogens, virulence factors and regulators have been characterized. Within EVs, these components could potentially impact disease outcomes by interacting with the host. From this perspective, we previously demonstrated that EVs from C. albicans could be taken up by and activate macrophages and dendritic cells to produce cytokines and express costimulatory molecules. Moreover, pre-treatment of Galleria mellonella larvae with fungal EVs protected the insects against a subsequent lethal infection with C. albicans yeasts. These data indicate that C. albicans EVs are multi-antigenic compartments that activate the innate immune system and could be exploited as vaccine formulations. Here we investigated whether immunization with C. albicans EVs induces a protective effect against murine candidiasis in immunosuppressed mice. Total and fungal antigen-specific serum IgG antibodies increased by 21 days after immunization, confirming the efficacy of the protocol. Vaccination decreased fungal burden in the liver, spleen and kidney of mice challenged with C. albicans. Splenic levels of cytokines indicated a lower inflammatory response in mice immunized with EVs when compared with EVs+Freund's adjuvant (ADJ). Higher levels of IL-12p70, TNFα and IFNγ were detected in mice vaccinated with EVs+ADJ, while IL-12p70, TGFβ, IL-4 and IL-10 were increased when no adjuvants were added. Full protection of lethally challenged mice was observed when EVs were administered, regardless the presence of adjuvant. Physical properties of the EVs were also investigated and EVs produced by C. albicans were relatively stable after storage at 4, -20 or -80 0 C, keeping their ability to activate dendritic cells and to protect G. mellonella against a lethal candidiasis. Our data suggest that fungal EVs could be a safe source of antigens to be exploited in vaccine formulations. This article is protected by copyright. All rights reserved.