Studying the Humoral Response against SARS-CoV-2 in Cuban COVID-19 Recovered Patients.

Understanding the immune response generated by SARS-CoV-2 is critical for assessing efficient therapeutic protocols and gaining insights into the durability of protective immunity. The current work was aimed at studying the specific humoral responses against SARS-CoV-2 in Cuban COVID-19 convalescents. We developed suitable tools and methods based on ELISA methodology, for supporting this evaluation. Here, we describe the development of an ELISA for the quantification of anti-RBD IgG titers in a large number of samples and a similar test in the presence of NH4SCN as chaotropic agent for estimating the RBD specific antibody avidity. Additionally, a simple and rapid ELISA based on antibody-mediated blockage of the binding RBD-ACE2 was implemented for detecting, as a surrogate of conventional test, the levels of anti-RBD inhibitory antibodies in convalescent sera. In a cohort of 273 unvaccinated convalescents, we identified higher anti-RBD IgG titer (1 : 1,330, p < 0.0001) and higher levels of inhibitory antibodies blocking RBD-ACE2 binding (1 : 216, p < 0.05) among those who had recovered from severe illness. Our results suggest that disease severity, and not demographic features such as age, sex, and skin color, is the main determinant of the magnitude and neutralizing ability of the anti-RBD antibody response. An additional paired longitudinal assessment in 14 symptomatic convalescents revealed a decline in the antiviral antibody response and the persistence of neutralizing antibodies for at least 4 months after the onset of symptoms. Overall, SARS-CoV-2 infection elicits different levels of antibody response according to disease severity that declines over time and can be monitored using our homemade serological assays.

Jamaican fruit bats mount a stable and highly neutralizing antibody response after bat influenza virus infection.

Antibodies are an essential component of the antiviral response in many species, but to date, there is no compelling evidence that bats are capable of eliciting a robust humoral immunity, including neutralizing antibodies. Here, we report that infection of Jamaican fruit bats with the bat influenza A virus H18N11 elicits a rapid and stable humoral immune response with a strong neutralizing capacity, associated with no detectable viral shedding after repeat challenge infection. Thus, the neutralizing antibody response of bats might play an important role in the bat immunity.

Detection of Neutralizing Antibodies in Serum Samples Using a SARS-CoV-2 Pseudotyped Virus Assay.

Conventional live virus research on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causal agent of coronavirus disease-19 (COVID-19), requires Biosafety Level 3 (BSL-3) facilities. SARS-CoV-2 pseudotyped viruses have emerged as valuable tools in virology, mimicking the entry process of the SARS-CoV-2 virus into human cells by expressing its spike glycoprotein in a surrogate system using recombinant plasmids. One significant application of this tool is in functional assays for the evaluation of neutralizing antibodies. Pseudotyped viruses have the advantage of being competent for only a single cycle of infection, providing better safety and versatility and allowing them to be studied in BSL-2 laboratories. Here, we describe three protocols for the detection of SARS-CoV-2 neutralizing antibodies through a pseudotyped virus assay. First, SARS-CoV-2 S pseudotyped viruses (PV SARS-CoV-2 S) are produced using a Moloney murine leukemia virus (MuLV) three-plasmid system. The plasmids are designed to express the GagPol packing proteins, enhanced green fluorescent protein (eGFP) as a readout system, and the SARS-CoV-2 S protein modified to remove the endoplasmic reticulum retention domain and to improve infection. Next, the internalization of PV SARS-CoV-2 S protein in human embryonic kidney 293T (HEK-293T) cells overexpressing angiotensin-converting enzyme 2 (HEK-293T-ACE2) is confirmed by fluorescence microscopy and quantified using flow cytometry. Finally, PV SARS-CoV-2 S is used to screen neutralizing antibodies in serum samples from convalescent COVID-19 patients; it can also be used for studying the cell entry mechanisms of different SARS-CoV-2 variants, evaluating antiviral agents, and designing vaccines. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Generation of PV SARS-CoV-2 S pseudotyped virus Basic Protocol 2: Assay of PV SARS-CoV-2 S internalization in target cells. Basic Protocol 3: Detection of neutralizing antibodies in serum samples.

Sputnik V-Induced Antibodies against SARS-CoV-2 Variants during the Dissemination of the Gamma Variant in Venezuela.

The COVID-19 pandemic was characterized by the emergence and succession of SARS-CoV-2 variants able to evade the antibody response induced by natural infection and vaccination. To evaluate the IgG reactivity and neutralizing capacity of the serum of individuals vaccinated with Sputnik V (105 volunteers vaccinated) against different viral variants. IgG reactivity to the Spike protein (S) was evaluated by ELISA. A plaque reduction neutralization test was performed using different viral variant isolates. At 42 days post-vaccination, the frequency of recognition and reactivity to the S protein of the Omicron variant was lower compared to that of the other variants. In general, a higher average neutralization titer was seen against the ancestral variant compared to the variants, especially Omicron. However, some sera exhibited a higher neutralization titer to the Gamma variant compared to the ancestral variant, suggesting unapparent exposure during the clinical trial. Antibodies induced by Sputnik V can recognize, persist, and neutralize SARS-CoV-2 variants, with Omicron being the one that best evades this response. These results represent a unique report on the humoral response induced by a globally lesser-studied vaccine in terms of efficacy and immune escape, offering insights into developing vaccines targeting unknown coronaviruses.

Influence of previous COVID-19 exposure and vaccine type (CoronaVac, ChAdOx1 nCov-19 or BNT162b2) on antibody and cytokine (Th1 or Th2) responses.

To achieve global herd immunity, widespread vaccination is the most effective strategy. Vaccines stimulate the immune system, generating cytokines and chemokines, isotype antibodies, and neutralizing antibodies; all these molecules collectively provide a more comprehensive characterization of the immune response post-vaccination. We conducted a longitudinal study in northwestern Mexico, involving 120 individuals before vaccination and after the first dose of the SARS-CoV-2 vaccine, and 46 individuals after their second dose. Our findings reveal that antibody levels stabilize over time; cytokine levels generally increase following the first dose but decrease after the second dose and higher than normal levels in IgG1 and IgG3 concentrations are present. Most of the innate cytokines determined in this study were higher after the first dose of the vaccine. Regardless of previous infection history, this finding suggests that the first dose of the vaccine is crucial and may stimulate immunity by enhancing the innate immune response. Conversely, increased levels of IL-4, indicative of a Th2 response, were found in individuals without prior exposure to the virus and in those vaccinated with CoronaVac. These results suggest that the immune response to COVID-19 vaccines is multi-faceted, with preexisting immunity potentiating a more robust innate response. Vaccine type plays a critical role, with genetic vaccines favoring a Th1 response and inactivated vaccines like CoronaVac skewing toward a Th2 profile.

Immunologic mediators profile in COVID-19 convalescence.

SARS-CoV-2 caused the pandemic situation experienced since the beginning of 2020, and many countries faced the rapid spread and severe form of the disease. Mechanisms of interaction between the virus and the host were observed during acute phase, but few data are available when related to immunity dynamics in convalescents. We conducted a longitudinal study, with 51 healthy donors and 62 COVID-19 convalescent patients, which these had a 2-month follow-up after symptoms recovery. Venous blood sample was obtained from all participants to measure blood count, subpopulations of monocytes, lymphocytes, natural killer cells and dendritic cells. Serum was used to measure cytokines, chemokines, growth factors, anti-N IgG and anti-S IgG/IgM antibodies. Statistic was performed by Kruskal-Wallis test, and linear regression with days post symptoms and antibody titers. All analysis had confidence interval of 95%. Less than 35% of convalescents were anti-S IgM+, while more than 80% were IgG+ in D30. Anti-N IgG decreased along time, with loss of seroreactivity of 13%. Eosinophil count played a distinct role on both antibodies during all study, and the convalescence was orchestrated by higher neutrophil-to-lymphocyte ratio and IL-15, but initial stages were marked by increase in myeloid DCs, B1 lymphocytes, inflammatory and patrolling monocytes, G-CSF and IL-2. Later convalescence seemed to change to cytotoxicity mediated by T lymphocytes, plasmacytoid DCs, VEGF, IL-9 and CXCL10. Anti-S IgG antibodies showed the longest perseverance and may be a better option for diagnosis. The inflammatory pattern is yet present on initial stage of convalescence, but quickly shifts to a reparative dynamic. Meanwhile eosinophils seem to play a role on anti-N levels in convalescence, although may not be the major causative agent. We must highlight the importance of immunological markers on acute clinical outcomes, but their comprehension to potentialize adaptive system must be explored to improve immunizations and further preventive policies.

Do immunosuppressive treatments influence immune responses against adenovirus-based COVID-19 vaccines in patients with multiple sclerosis? An Argentine multicenter study.

Introduction: There are no reports in LATAM related to longitudinal humoral and cellular response to adenovirus based COVID-19 vaccines in people with Multiple Sclerosis (pwMS) under different disease modifying therapies (DMTs) and neutralization of the Omicron and Wuhan variants of SARS-COV-2. Methods: IgG anti- SARS-COV-2 spike titer were measured in a cohort of 101 pwMS under fingolimod, dimethyl fumarate, cladribine and antiCD20, as well as 28 healthy controls (HC) were measured 6 weeks after vaccination with 2nd dose (Sputnik V or AZD1222) and 3nd dose (homologous or heterologous schedule). Neutralizing capacity was against Omicron (BA.1) and Wuhan (D614G) variants and pseudotyped particles and Cellular response were analyzed. Results: Multivariate regression analysis showed anti-cd20 (ß= -,349, 95% CI: -3655.6 - -369.01, p=0.017) and fingolimod (ß=-,399, 95% CI: -3363.8 - -250.9, p=0.023) treatments as an independent factor associated with low antibody response (r2 adjusted=0.157). After the 2nd dose we found a correlation between total and neutralizing titers against D614G (rho=0.6; p<0.001; slope 0.8, 95%CI:0.4-1.3), with no differences between DMTs. Neutralization capacity was lower for BA.1 (slope 0.3, 95%CI:0.1-0.4). After the 3rd dose, neutralization of BA.1 improved (slope: 0.9 95%CI:0.6-1.2), without differences between DMTs. A fraction of pwMS generated anti-Spike CD4+ and CD8+ T cell response. In contrast, pwMS under antiCD20 generated CD8+TNF+IL2+ response without differences with HC, even in the absence of humoral response. The 3rd dose significantly increased the neutralization against the Omicron, as observed in the immunocompetent population. Discussion: Findings regarding humoral and cellular response are consistent with previous reports.

Enhanced Assessment of Cross-Reactive Antigenic Determinants within the Spike Protein.

Despite successful vaccination efforts, the emergence of new SARS-CoV-2 variants poses ongoing challenges to control COVID-19. Understanding humoral responses regarding SARS-CoV-2 infections and their impact is crucial for developing future vaccines that are effective worldwide. Here, we identified 41 immunodominant linear B-cell epitopes in its spike glycoprotein with an SPOT synthesis peptide array probed with a pool of serum from hospitalized COVID-19 patients. The bioinformatics showed a restricted set of epitopes unique to SARS-CoV-2 compared to other coronavirus family members. Potential crosstalk was also detected with Dengue virus (DENV), which was confirmed by screening individuals infected with DENV before the COVID-19 pandemic in a commercial ELISA for anti-SARS-CoV-2 antibodies. A high-resolution evaluation of antibody reactivity against peptides representing epitopes in the spike protein identified ten sequences in the NTD, RBD, and S2 domains. Functionally, antibody-dependent enhancement (ADE) in SARS-CoV-2 infections of monocytes was observed in vitro with pre-pandemic Dengue-positive sera. A significant increase in viral load was measured compared to that of the controls, with no detectable neutralization or considerable cell death, suggesting its role in viral entry. Cross-reactivity against peptides from spike proteins was observed for the pre-pandemic sera. This study highlights the importance of identifying specific epitopes generated during the humoral response to a pathogenic infection to understand the potential interplay of previous and future infections on diseases and their impact on vaccinations and immunodiagnostics.

An unexpected IgE anti-receptor binding domain response following natural infection and different types of SARS-CoV-2 vaccines.

Humoral response to SARS-CoV-2 has been studied, predominantly the classical IgG and its subclasses. Although IgE antibodies are typically specific to allergens or parasites, a few reports describe their production in response to SARS-CoV-2 and other viruses. Here, we investigated IgE specific to receptor binding domain (RBD) of SARS-CoV-2 in a Brazilian cohort following natural infection and vaccination. Samples from 59 volunteers were assessed after infection (COVID-19), primary immunization with vectored (ChAdOx1) or inactivated (CoronaVac) vaccines, and booster immunization with mRNA (BNT162b2) vaccine. Natural COVID-19 induced IgE, but vaccination increased its levels. Subjects vaccinated with two doses of ChAdOx1 exhibited a more robust response than those immunized with two doses of CoronaVac; however, after boosting with BNT162b2, all groups presented similar IgE levels. IgE showed intermediate-to-high avidity, especially after the booster vaccine. We also found IgG4 antibodies, mainly after the booster, and they moderately correlated with IgE. ELISA results were confirmed by control assays, using IgG depletion by protein G and lack of reactivity with heterologous antigen. In our cohort, no clinical data could be associated with the IgE response. We advocate for further research on IgE and its role in viral immunity, extending beyond allergies and parasitic infections.

COVID-19 IgG seropositivity and its determinants in occupational groups of varying infection risks in two Andean cities of Ecuador before mass vaccination.

BACKGROUND: The COVID-19 pandemic has caused over 68.7 million infections and 1.35 million deaths in South America. There are limited data on SARS-CoV-2 seropositivity and its determinants from Andean countries prior to mass vaccinations against COVID-19. OBJECTIVE: To estimate SARS-CoV-2 seropositivity and its determinants before vaccination in occupational groups of adults presumed to have different levels of exposure and associations with potential symptomatology. METHODS: We measured seropositivity of anti-SARS-CoV-2 IgG antibodies in a cross-sectional study of vaccine-naïve adults aged 18 years and older, recruited within three occupational risk groups (defined as low [LR], moderate [MR], and high [HR]) between January and September 2021 in two Andean cities in Ecuador. Associations with risk factors were estimated using logistic regression. RESULTS: In a sample of 882 adults, IgG seropositivity for the three different occupational risk groups was 39.9% (CI 95% 35.3-44.6), 74.6% (CI 95% 66.4-81.4), and 39.0% (CI 95% 34.0-44.4) for the HR, MR, and LR groups, respectively. History of an illness with loss of taste and/or smell was significantly associated with seropositivity in all occupational groups, with adjusted ORs of 14.31 (95%CI, 5.83-35.12; p<0.001), 14.34 (95%CI 3.01-68.42; p<0.001), and 8.79 (95%CI 2.69-28.72; p<0.001), for the HR, MR, and LR groups, respectively; while fever was significant for the LR group with an adjusted OR of 1.24 (95%CI, 1.11-4.57; p = 0.025) and myalgia for the HR group with an adjusted OR of 2.07 (95%CI, 1.13-3.81; p = 0.019). CONCLUSION: Notable proportions of seropositivity were seen in all occupational groups between January and September 2021 prior to mass vaccination. Loss of taste and/or smell was strongly associated with presence of anti-SARS-CoV-2 IgG antibodies irrespective of presumed occupational exposure risk.

Co-administration of recombinant BCG and SARS-CoV-2 proteins leads to robust antiviral immunity.

SARS-CoV-2 is the causative virus of COVID-19, which has been responsible for millions of deaths worldwide since its discovery. After its emergence, several variants have been identified that challenge the efficacy of the available vaccines. Previously, we generated and evaluated a vaccine based on a recombinant Bacillus Calmette-Guérin (rBCG) expressing the nucleoprotein (N) of SARS-CoV-2 (rBCG-N-SARS-CoV-2). This protein is a highly immunogenic antigen and well conserved among variants. Here, we tested the administration of this vaccine with recombinant N and viral Spike proteins (S), or Receptor Binding Domain (RBD-Omicron variant), plus a booster with the recombinant proteins only, as a novel and effective strategy to protect against SARS-CoV-2 variants. METHODS: BALB/c mice were immunized with rBCG-N-SARS-CoV-2 and recombinant SARS-CoV-2 proteins in Alum adjuvant, followed by a booster with recombinant proteins to assess the safety and virus-specific cellular and humoral immune responses against SARS-CoV-2 antigens. RESULTS: Immunization with rBCG-N-SARS-CoV-2 + recombinant proteins as a vaccine was safe and promoted the activation of CD4+ and CD8+ T cells that recognize SARS-CoV-2 N, S, and RBD antigens. These cells were able to secrete cytokines with an antiviral profile. This immunization strategy also induced robust titers of specific antibodies against N, S, and RBD and neutralizing antibodies of SARS-CoV-2. CONCLUSIONS: Co-administration of the rBCG-N-SARS-CoV-2 vaccine with recombinant SARS-CoV-2 proteins could be an effective alternative to control particular SARS-CoV-2 variants. Due to its safety and capacity to induce virus-specific immune responses, we believe the rBCG-N-SARS-CoV-2 + Proteins vaccine could be an attractive candidate to protect against this virus, especially in newborns.

Recombinant Subunit Vaccine Candidate against the Bovine Viral Diarrhea Virus.

Multivalent live-attenuated or inactivated vaccines are often used to control the bovine viral diarrhea disease (BVD). Still, they retain inherent disadvantages and do not provide the expected protection. This study developed a new vaccine prototype, including the external segment of the E2 viral protein from five different subgenotypes selected after a massive screening. The E2 proteins of every subgenotype (1aE2, 1bE2, 1cE2, 1dE2, and 1eE2) were produced in mammalian cells and purified by IMAC. An equimolar mixture of E2 proteins formulated in an oil-in-water adjuvant made up the vaccine candidate, inducing a high humoral response at 50, 100, and 150 µg doses in sheep. A similar immune response was observed in bovines at 50 µg. The cellular response showed a significant increase in the transcript levels of relevant Th1 cytokines, while those corresponding to the Th2 cytokine IL-4 and the negative control were similar. High levels of neutralizing antibodies against the subgenotype BVDV1a demonstrated the effectiveness of our vaccine candidate, similar to that observed in the sera of animals vaccinated with the commercial vaccine. These results suggest that our vaccine prototype could become an effective recombinant vaccine against the BVD.

Genetic, Clinical, Epidemiological, and Immunological Profiling of IgG Response Duration after SARS-CoV-2 Infection.

The IgG response against SARS-CoV-2 infection can persist for over six months (long response; LR). However, among 30% of those infected, the duration can be as short as three months or less (short response; SR). The present study assembled serological data on the anti-SARS-CoV-2 IgG response duration of two previous studies and integrated these results with the plasmatic cytokine levels and genetic profile of 10 immune-relevant SNPs that were also previously published, along with the plasmatic total IgG, IgA, and IgM levels, allowing for the genetic, clinical, immunological, and epidemiological aspects of the post-COVID-19 IgG response duration to be understood. The SR was associated with previous mild acute COVID-19 and with an SNP (rs2228145) in IL6R related to low gene expression. Additionally, among the SR subgroup, no statistically significant Spearman correlations were observed between the plasma levels of IL-17A and the Th17 regulatory cytokines IFN-γ (rs = 0.2399; p = 0.1043), IL-4 (rs = 0.0273; p = 0.8554), and IL-2 (rs = 0.2204; p = 0.1365), while among the LR subgroup, weaker but statistically significant Spearman correlations were observed between the plasma levels of IL-17A and IFN-γ (rs = 0.3873; p = 0.0016), IL-4 (rs = 0.2671; p = 0.0328), and IL-2 (rs = 0.3959; p = 0.0012). These results suggest that the Th17 response mediated by the IL-6 pathway has a role in the prolonged IgG response to SARS-CoV-2 infection.

4th booster-dose SARS-CoV-2 heterologous and homologous vaccination in rheumatological patients.

Objective: to evaluate the immune response to the SARS-CoV-2 vaccines in adults with immune-mediated rheumatic diseases (IMRDs) in comparison to healthy individuals, observed 1-20 weeks following the fourth vaccine dose. Additionally, to evaluate the impact of immunosuppressive therapies, vaccination schedules, the time interval between vaccination and sample collection on the vaccine's immune response. Methods: We designed a longitudinal observational study conducted at the rheumatology department of Hospital de Copiapó. Neutralizing antibodies (Nabs) titers against the Wuhan and Omicron variant were analyzed between 1-20 weeks after administration of the fourth dose of the SARS-CoV-2 vaccine to 341 participants (218 IMRD patients and 123 healthy controls). 218 IMRD patients with rheumatoid arthritis (RA), psoriatic arthritis (PsA), ankylosing spondylitis (AS), systemic lupus erythematosus (SLE), systemic vasculitis (VS) and systemic scleroderma (SS) were analyzed. Results: Performing a comparison between the variants, Wuhan vs Omicron, we noticed that there were significant differences (p<0.05) in the level of the ID50, both for healthy controls and for patients with IMRDs. The humoral response of patients with IMRDs is significantly lower compared to healthy controls for the Omicron variant of SARS-CoV-2 (p = 0.0015). The humoral response of patients with IMRDs decreases significantly when the time interval between vaccination and sample collection is greater than 35 days. This difference was observed in the response, both for the Wuhan variant and for the Omicron variant. Conclusion: The IMRDs patients, the humoral response variation in the SARS-CoV-2 vaccine depends on doses and type of vaccine administered, the humoral response times and the treatment that these patients are receiving.

Efficacy, immunogenicity and safety of CoronaVac® in children and adolescents aged 6 months to 17 years: a multicenter, randomized, double-blind, placebo-controlled phase III clinical trial.

Safe and effective vaccines against COVID-19 for children and adolescents are needed. This international multicenter, randomized, double-blind, placebo-controlled, phase III clinical trial assessed the efficacy, immunogenicity, and safety of CoronaVac® in children and adolescents (NCT04992260). The study was carried out in Chile, South Africa, Malaysia, and the Philippines. The enrollment ran from September 10, 2021 to March 25, 2022. For efficacy assessment, the median follow-up duration from 14 days after the second dose was 169 days. A total of 11,349 subjects were enrolled. Two 3-µg injections of CoronaVac® or placebo were given 28 days apart. The primary endpoint was the efficacy of the CoronaVac®. The secondary endpoints were the immunogenicity and safety. The vaccine efficacy was 21.02% (95% CI: 1.65, 36.67). The level of neutralizing antibody in the vaccine group was significantly higher than that in the placebo group (GMT: 390.80 vs. 62.20, P <0.0001). Most adverse reactions were mild or moderate. All the severe adverse events were determined to be unrelated to the investigational products. In conclusion, in the Omicron-dominate period, a two-dose schedule of 3 µg CoronaVac® was found to be safe and immunogenic, and showed potential against symptomatic COVID-19 in healthy children and adolescents.

Seroprevalence trends of anti-SARS-CoV-2 antibodies in the adult population of the São Paulo Municipality, Brazil: Results from seven serosurveys from June 2020 to April 2022. The SoroEpi MSP Study.

BACKGROUND: Sequential population-based household serosurveys of SARS-CoV-2 covering the COVID-19 pre- and post-vaccination periods are scarce in Brazil. This study investigated seropositivity trends in the municipality of São Paulo. METHODS: We conducted seven cross-sectional surveys of adult population-representative samples between June 2020 and April 2022. The study design included probabilistic sampling, test for SARS-CoV-2 antibodies using the Roche Elecsys anti-nucleocapsid assay, and statistical adjustments for population demographics and non-response. The weighted seroprevalences with 95% confidence intervals (CI) were estimated by sex, age group, race, schooling, and mean income study strata. Time trends in seropositivity were assessed using the Joinpoint model. We compared infection-induced seroprevalences with COVID-19 reported cases in the pre-vaccination period. RESULTS: The study sample comprised 8,134 adults. The overall SARS-CoV-2 seroprevalence increased from 11.4% (95%CI: 9.2-13.6) in June 2020 to 24.9% (95%CI: 21.0-28.7) in January 2021; from 38.1% (95%CI: 34.3-41.9) in April 2021 to 77.7% (95%CI: 74.4-81.0) in April 2022. The prevalence over time was higher in the subgroup 18-39 years old than in the older groups from Survey 3 onwards. The self-declared Black or mixed (Pardo) group showed a higher prevalence in all surveys compared to the White group. Monthly prevalence rose steeply from January 2021 onwards, particularly among those aged 60 years or older. The infection-to-case ratios ranged from 8.9 in June 2020 to 4.3 in January 2021. CONCLUSIONS: The overall seroprevalence rose significantly over time and with age and race subgroup variations. Increases in the 60 years or older age and the White groups were faster than in younger ages and Black or mixed (Pardo) race groups in the post-vaccination period. Our data may add to the understanding of the complex and changing population dynamics of the SARS-CoV-2 infection, including the impact of vaccination strategies and the modelling of future epidemiological scenarios.

Precision arbovirus serology with a pan-arbovirus peptidome.

Arthropod-borne viruses represent a crucial public health threat. Current arboviral serology assays are either labor intensive or incapable of distinguishing closely related viruses, and many zoonotic arboviruses that may transition to humans lack any serologic assays. In this study, we present a programmable phage display platform, ArboScan, that evaluates antibody binding to overlapping peptides that represent the proteomes of 691 human and zoonotic arboviruses. We confirm that ArboScan provides detailed antibody binding information from animal sera, human sera, and an arthropod blood meal. ArboScan identifies distinguishing features of antibody responses based on exposure history in a Colombian cohort of Zika patients. Finally, ArboScan details epitope level information that rapidly identifies candidate epitopes with potential protective significance. ArboScan thus represents a resource for characterizing human and animal arbovirus antibody responses at cohort scale.

Antibody levels against respiratory syncytial virus fusion protein conformations and lack of association with life-threatening infection in previously healthy infants.

BACKGROUND: Humoral immune response against the pre-fusion (pre-F) conformation of respiratory syncytial virus (RSV) F protein has been proposed to play a protective role against infection. An RSV pre-F maternal vaccine has been recently approved in several countries to protect young infants against RSV. We aimed to assess serum IgG titers against the pre-F and post-F conformations of RSV F protein and their association with life-threatening RSV disease (LTD) in previously healthy infants. METHODS: A prospective cohort study including hospitalized infants <12 months with a first RSV infection was conducted during 2017-2019. Patients with LTD required intensive care and mechanical respiratory assistance. RSV pre-F exclusive and post-F antibody responses were determined by post-F competition and non-competition immunoassays, respectively, and neutralizing activity was measured by plaque reduction neutralization test. RESULTS: Fifty-eight patients were included; the median age was 3.5 months and 41 % were females. Fifteen patients developed LTD. RSV F-specific antibody titers positively correlated with neutralizing antibody titers in acute and convalescent phases but, importantly, they did not associate with LTD. Acute RSV pre-F exclusive and post-F IgG titers negatively correlated with patient age (P = 0.0007 and P < 0.0001), while a positive correlation was observed between the fold changes in RSV F-specific antibody titers between convalescent and acute phase and patient age (P = 0.0014 and P < 0.0001). Infants ≤2 months exhibited significantly lower fold-changes in RSV F-specific and neutralizing antibody titers between convalescence and acute phase than older infants. Additionally, acute RSV antibody titers showed no correlation with nasal RSV load and, furthermore, nasal viral load was not associated with the development of LTD. CONCLUSIONS: This study highlights that protection against life-threatening RSV disease is not necessarily antibody-dependent. Further characterization of the immune response against RSV and its role in protection against severe disease is important for the development of the safest possible preventive strategies.

ATP-P2X7R pathway activation limits the Tfh cell compartment during pediatric RSV infection.

Background: Follicular helper T cells (Tfh) are pivotal in B cell responses. Activation of the purinergic receptor P2X7 on Tfh cells regulates their activity. We investigated the ATP-P2X7R axis in circulating Tfh (cTfh) cells during Respiratory Syncytial Virus (RSV) infection. Methods: We analyzed two cohorts: children with RSV infection (moderate, n=30; severe, n=21) and healthy children (n=23). We utilized ELISA to quantify the levels of PreF RSV protein-specific IgG antibodies, IL-21 cytokine, and soluble P2X7R (sP2X7R) in both plasma and nasopharyngeal aspirates (NPA). Additionally, luminometry was employed to determine ATP levels in plasma, NPA and supernatant culture. The frequency of cTfh cells, P2X7R expression, and plasmablasts were assessed by flow cytometry. To evaluate apoptosis, proliferation, and IL-21 production by cTfh cells, we cultured PBMCs in the presence of Bz-ATP and/or P2X7R antagonist (KN-62) and a flow cytometry analysis was performed. Results: In children with severe RSV disease, we observed diminished titers of neutralizing anti-PreF IgG antibodies. Additionally, severe infections, compared to moderate cases, were associated with fewer cTfh cells and reduced plasma levels of IL-21. Our investigation revealed dysregulation in the ATP-P2X7R pathway during RSV infection. This was characterized by elevated ATP levels in both plasma and NPA samples, increased expression of P2X7R on cTfh cells, lower levels of sP2X7R, and heightened ATP release from PBMCs upon stimulation, particularly evident in severe cases. Importantly, ATP exposure decreased cTfh proliferative response and IL-21 production, while promoting their apoptosis. The P2X7R antagonist KN-62 mitigated these effects. Furthermore, disease severity positively correlated with ATP levels in plasma and NPA samples and inversely correlated with cTfh frequency. Conclusion: Our findings indicate that activation of the ATP-P2X7R pathway during RSV infection may contribute to limiting the cTfh cell compartment by promoting cell death and dysfunction, ultimately leading to increased disease severity.

The seroconversion history to SARS-CoV-2 in Indigenous people from Brazil - the interplay between exposure, vaccination, and tuberculosis.

The COVID-19 pandemic caused a significant loss of human lives and a worldwide decline in quality of life. Although our understanding of the pandemic has improved significantly since the beginning, the natural history of COVID-19 and its impacts on under-represented populations, such as Indigenous people from America, remain largely unknown. We performed a retrospective serological survey with two Brazilian Indigenous populations (n=624), Tupiniquim and Guarani-Mbyá. Samples were collected between September 2020 and July 2021: a period comprising the dissemination of SARS-CoV-2 variants and the beginning of COVID-19 vaccination in Brazil. Seroconversions against S and N antigens were assessed using three different commercially available ELISA kits. Samples were also used to assess the prevalence of tuberculosis (TB) in the same population (n=529). Seroconversion against SARS-CoV-2 antigens was considered positive if at least one of the three ELISA kits detected levels of specific antibodies above the threshold specified by the manufacturer. In this sense, we report 56.0% (n=349/623) of seroconverted individuals. Relative seroconversion peaked after introduction of the Coronavac vaccine in February 2021. Vaccination increased the production of anti-S IgG from 3.9% to 48.6%. Our results also indicated that 11.0% (n=46/417) of all individuals were positive for TB. Seroconversion to SARS-CoV-2 was similar between individuals with positive tuberculosis test results to those with negative test results. Most vaccinated individuals seroconverted to SARS-CoV-2, indicating that Coronavac may be as protective in individuals from these indigenous groups as observed in the general Brazilian population. COVID-19 severity was minimal regardless of incomplete vaccine coverage, suggesting that vaccination may not be the only factor protecting individuals from severe COVID-19. Tuberculosis is highly prevalent and not associated with increased seroconversion to SARS-CoV-2.