Distinct T cell responsiveness to different COVID-19 vaccines and cross-reactivity to SARS-CoV-2 variants with age and CMV status.

Introduction: Accumulating evidence indicates the importance of T cell immunity in vaccination-induced protection against severe COVID-19 disease, especially against SARS-CoV-2 Variants-of-Concern (VOCs) that more readily escape from recognition by neutralizing antibodies. However, there is limited knowledge on the T cell responses across different age groups and the impact of CMV status after primary and booster vaccination with different vaccine combinations. Moreover, it remains unclear whether age has an effect on the ability of T cells to cross-react against VOCs. Methods: Therefore, we interrogated the Spike-specific T cell responses in healthy adults of the Dutch population across different ages, whom received different vaccine types for the primary series and/or booster vaccination, using IFNÉ£ ELISpot. Cells were stimulated with overlapping peptide pools of the ancestral Spike protein and different VOCs. Results: Robust Spike-specific T cell responses were detected in the vast majority of participants upon the primary vaccination series, regardless of the vaccine type (i.e. BNT162b2, mRNA-1273, ChAdOx1 nCoV-19, or Ad26.COV2.S). Clearly, in the 70+ age group, responses were overall lower and showed more variation compared to younger age groups. Only in CMV-seropositive older adults (>70y) there was a significant inverse relation of age with T cell responses. Although T cell responses increased in all age groups after booster vaccination, Spike-specific T cell frequencies remained lower in the 70+ age group. Regardless of age or CMV status, primary mRNA-1273 vaccination followed by BNT162b2 booster vaccination showed limited booster effect compared to the BNT162b2/BNT162b2 or BNT162b2/mRNA-1273 primary-booster regimen. A modest reduction in cross-reactivity to the Alpha, Delta and Omicron BA.1, but not the Beta or Gamma variant, was observed after primary vaccination. Discussion: Together, this study shows that age, CMV status, but also the primary-booster vaccination regimen influence the height of the vaccination-induced Spike-specific T cell response, but did not impact the VOC cross-reactivity.

mRNA-LNP HIV-1 trimer boosters elicit precursors to broad neutralizing antibodies.

Germline-targeting (GT) HIV vaccine strategies are predicated on deriving broadly neutralizing antibodies (bnAbs) through multiple boost immunogens. However, as the recruitment of memory B cells (MBCs) to germinal centers (GCs) is inefficient and may be derailed by serum antibody-induced epitope masking, driving further B cell receptor (BCR) modification in GC-experienced B cells after boosting poses a challenge. Using humanized immunoglobulin knockin mice, we found that GT protein trimer immunogen N332-GT5 could prime inferred-germline precursors to the V3-glycan-targeted bnAb BG18 and that B cells primed by N332-GT5 were effectively boosted by either of two novel protein immunogens designed to have minimum cross-reactivity with the off-target V1-binding responses. The delivery of the prime and boost immunogens as messenger RNA lipid nanoparticles (mRNA-LNPs) generated long-lasting GCs, somatic hypermutation, and affinity maturation and may be an effective tool in HIV vaccine development.

A potent Henipavirus cross-neutralizing antibody reveals a dynamic fusion-triggering pattern of the G-tetramer.

The Hendra and Nipah viruses (HNVs) are highly pathogenic pathogens without approved interventions for human use. In addition, the interaction pattern between the attachment (G) and fusion (F) glycoproteins required for virus entry remains unclear. Here, we isolate a panel of Macaca-derived G-specific antibodies that cross-neutralize HNVs via multiple mechanisms. The most potent antibody, 1E5, confers adequate protection against the Nipah virus challenge in female hamsters. Crystallography demonstrates that 1E5 has a highly similar binding pattern to the receptor. In cryo-electron microscopy studies, the tendency of 1E5 to bind to the upper or lower heads results in two distinct quaternary structures of G. Furthermore, we identify the extended outer loop ß1S2-ß1S3 of G and two pockets on the apical region of fusion (F) glycoprotein as the essential sites for G-F interactions. This work highlights promising drug candidates against HNVs and contributes deeper insights into the viruses.

GNUV201, a novel human/mouse cross-reactive and low pH-selective anti-PD-1 monoclonal antibody for cancer immunotherapy.

BACKGROUND: Several PD-1 antibodies approved as anti-cancer therapies work by blocking the interaction of PD-1 with its ligand PD-L1, thus restoring anti-cancer T cell activities. These PD-1 antibodies lack inter-species cross-reactivity, necessitating surrogate antibodies for preclinical studies, which may limit the predictability and translatability of the studies. RESULTS: To overcome this limitation, we have developed an inter-species cross-reactive PD-1 antibody, GNUV201, by utilizing an enhanced diversity mouse platform (SHINE MOUSE™). GNUV201 equally binds to human PD-1 and mouse PD-1, equally inhibits the binding of human PD-1/PD-L1 and mouse PD-1/PD-L1, and effectively suppresses tumor growth in syngeneic mouse models. The epitope of GNUV201 mapped to the "FG loop" of hPD-1, distinct from those of Keytruda® ("C'D loop") and Opdivo® (N-term). Notably, the structural feature where the protruding epitope loop fits into GNUV201's binding pocket supports the enhanced binding affinity due to slower dissociation (8.7 times slower than Keytruda®). Furthermore, GNUV201 shows a stronger binding affinity at pH 6.0 (5.6 times strong than at pH 7.4), which mimics the hypoxic and acidic tumor microenvironment (TME). This phenomenon is not observed with marketed antibodies (Keytruda®, Opdivo®), implying that GNUV201 achieves more selective binding to and better occupancy on PD-1 in the TME. CONCLUSIONS: In summary, GNUV201 exhibited enhanced affinity for PD-1 with slow dissociation and preferential binding in TME-mimicking low pH. Human/monkey/mouse inter-species cross-reactivity of GNUV201 could enable more predictable and translatable efficacy and toxicity preclinical studies. These results suggest that GNUV201 could be an ideal antibody candidate for anti-cancer drug development.

Trimeric Bet v 1-specific nanobodies cause strong suppression of IgE binding.

Background: Around 20% of the population in Northern and Central Europe is affected by birch pollen allergy, with the major birch pollen allergen Bet v 1 as the main elicitor of allergic reactions. Together with its cross-reactive allergens from related trees and foods, Bet v 1 causes an impaired quality of life. Hence, new treatment strategies were elaborated, demonstrating the effectiveness of blocking IgG antibodies on Bet v 1-induced IgE-mediated reactions. A recent study provided evidence for the first time that Bet v 1-specific nanobodies reduce patients´ IgE binding to Bet v 1. In order to increase the potential to outcompete IgE recognition of Bet v 1 and to foster cross-reactivity and cross-protection, we developed Bet v 1-specific nanobody trimers and evaluated their capacity to suppress polyclonal IgE binding to corresponding allergens and allergen-induced basophil degranulation. Methods: Nanobody trimers were engineered by adding isoleucine zippers, thus enabling trimeric formation. Trimers were analyzed for their cross-reactivity, binding kinetics to Bet v 1, and related allergens, and patients' IgE inhibition potential. Finally, their efficacy to prevent basophil degranulation was investigated. Results: Trimers showed enhanced recognition of cross-reactive allergens and increased efficiency to reduce IgE-allergen binding compared to nanobody monomers. Furthermore, trimers displayed slow dissociation rates from allergens and suppressed allergen-induced mediator release. Conclusion: We generated high-affine nanobody trimers that target Bet v 1 and related allergens. Trimers blocked IgE-allergen interaction by competing with IgE for allergen binding. They inhibited IgE-mediated release of biological mediators, demonstrating a promising potential to prevent allergic reactions caused by Bet v 1 and relatives.

Structure-based epitope prediction and assessment of cross-reactivity of Myrmecia pilosula venom-specific IgE and recombinant Sol g proteins (Solenopsis geminata).

The global distribution of tropical fire ants (Solenopsis geminata) raises concerns about anaphylaxis and serious medical issues in numerous countries. This investigation focused on the cross-reactivity of allergen-specific IgE antibodies between S. geminata and Myrmecia pilosula (Jack Jumper ant) venom proteins due to the potential emergence of cross-reactive allergies in the future. Antibody epitope analysis unveiled one predominant conformational epitope on Sol g 1.1 (PI score of 0.989), followed by Sol g 2.2, Sol g 4.1, and Sol g 3.1. Additionally, Pilosulin 1 showed high allergenic potential (PI score of 0.94), with Pilosulin 5a (PI score of 0.797) leading in B-cell epitopes. The sequence analysis indicated that Sol g 2.2 and Sol g 4.1 pose a high risk of cross-reactivity with Pilosulins 4.1a and 5a. Furthermore, the cross-reactivity of recombinant Sol g proteins with M. pilosula-specific IgE antibodies from 41 patients revealed high cross-reactivity for r-Sol g 3.1 (58.53%) and r-Sol g 4.1 (43.90%), followed by r-Sol g 2.2 (26.82%), and r-Sol g 1.1 (9.75%). Therefore, this study demonstrates cross-reactivity (85.36%) between S. geminata and M. pilosula, highlighting the allergenic risk. Understanding these reactions is vital for the prevention of severe allergic reactions, especially in individuals with pre-existing Jumper Jack ant allergy, informing future management strategies.

Comparison of Five Serological Methods for the Detection of West Nile Virus Antibodies.

The West Nile Virus (WNV), a member of the family Flaviviridae, is an emerging mosquito-borne flavivirus causing potentially severe infections in humans and animals involving the central nervous system (CNS). Due to its emerging tendency, WNV now occurs in many areas where other flaviviruses are co-occurring. Cross-reactive antibodies with flavivirus infections or vaccination (e.g., tick-borne encephalitis virus (TBEV), Usutu virus (USUV), yellow fever virus (YFV), dengue virus (DENV), Japanese encephalitis virus (JEV)) therefore remain a major challenge in diagnosing flavivirus infections. Virus neutralization tests are considered as reference tests for the detection of specific flavivirus antibodies, but are elaborate, time-consuming and need biosafety level 3 facilities. A simple and straightforward assay for the differentiation and detection of specific WNV IgG antibodies for the routine laboratory is urgently needed. In this study, we compared two commercially available enzyme-linked immunosorbent assays (anti-IgG WNV ELISA and anti-NS1-IgG WNV), a commercially available indirect immunofluorescence assay, and a newly developed in-house ELISA for the detection of WNV-NS1-IgG antibodies. All four tests were compared to an in-house NT to determine both the sensitivity and specificity of the four test systems. None of the assays could match the specificity of the NT, although the two NS1-IgG based ELISAs were very close to the specificity of the NT at 97.3% and 94.6%. The in-house WNV-NS1-IgG ELISA had the best performance regarding sensitivity and specificity. The specificities of the ELISA assays and the indirect immunofluorescence assays could not meet the necessary specificity and/or sensitivity.

Animal-derived food allergen: A review on the available crystal structure and new insights into structural epitope.

Immunoglobulin E (IgE)-mediated food allergy is a rapidly growing public health problem. The interaction between allergens and IgE is at the core of the allergic response. One of the best ways to understand this interaction is through structural characterization. This review focuses on animal-derived food allergens, overviews allergen structures determined by X-ray crystallography, presents an update on IgE conformational epitopes, and explores the structural features of these epitopes. The structural determinants of allergenicity and cross-reactivity are also discussed. Animal-derived food allergens are classified into limited protein families according to structural features, with the calcium-binding protein and actin-binding protein families dominating. Progress in epitope characterization has provided useful information on the structural properties of the IgE recognition region. The data reveals that epitopes are located in relatively protruding areas with negative surface electrostatic potential. Ligand binding and disulfide bonds are two intrinsic characteristics that influence protein structure and impact allergenicity. Shared structures, local motifs, and shared epitopes are factors that lead to cross-reactivity. The structural properties of epitope regions and structural determinants of allergenicity and cross-reactivity may provide directions for the prevention, diagnosis, and treatment of food allergies. Experimentally determined structure, especially that of antigen-antibody complexes, remains limited, and the identification of epitopes continues to be a bottleneck in the study of animal-derived food allergens. A combination of traditional immunological techniques and emerging bioinformatics technology will revolutionize how protein interactions are characterized.

Seroprevalence of the novel swine acute diarrhea syndrome coronavirus in China assessed by enzyme-linked immunosorbent assay.

The endemic outbreak of SADS-CoV has resulted in economic losses and potentially threatened the safety of China's pig industry. The molecular epidemiology of SADS-CoV in pig herds has been investigated in many provinces in China. However, there are no data over a long-time span, and there is a lack of extensive serological surveys to assess the prevalence of SADS-CoV in Chinese swine herds since the discovery of SADS-CoV. In this study, an indirect anti-SADS-CoV IgG enzyme-linked immunosorbent assay (ELISA) based on the SADS-CoV S1 protein was established to investigate the seroprevalence of SADS-CoV in Chinese swine herds. Cross-reactivity assays, indirect immunofluorescence, and western blotting assays showed that the developed ELISA had excellent SADS-CoV specificity. In total, 12,978 pig serum samples from 29 provinces/municipalities/autonomous regions in China were tested from 2022 to 2023. The results showed that the general seroprevalence of SADS-CoV in China was 59.97%, with seroprevalence ranging from 16.7% to 77.12% in different provinces and from 42.61% to 68.45% in different months. SADS-CoV is widely prevalent in China, and its seroprevalence was higher in Northeast China, North China, and Central China than in other regions. Among the four seasons, the prevalence of SADS-CoV was the highest in spring and the lowest in autumn. The results of this study provide the general seroprevalence profile of SADS-CoV in China, facilitating the understanding of the prevalence of SADS-CoV in pigs. More importantly, this study is beneficial in formulating preventive and control measures for SADS-CoV and may provide directions for vaccine development.

Trivalent mRNA vaccine-candidate against seasonal flu with cross-specific humoral immune response.

Seasonal influenza remains a serious global health problem, leading to high mortality rates among the elderly and individuals with comorbidities. Vaccination is generally accepted as the most effective strategy for influenza prevention. While current influenza vaccines are effective, they still have limitations, including narrow specificity for certain serological variants, which may result in a mismatch between vaccine antigens and circulating strains. Additionally, the rapid variability of the virus poses challenges in providing extended protection beyond a single season. Therefore, mRNA technology is particularly promising for influenza prevention, as it enables the rapid development of multivalent vaccines and allows for quick updates of their antigenic composition. mRNA vaccines have already proven successful in preventing COVID-19 by eliciting rapid cellular and humoral immune responses. In this study, we present the development of a trivalent mRNA vaccine candidate, evaluate its immunogenicity using the hemagglutination inhibition assay, ELISA, and assess its efficacy in animals. We demonstrate the higher immunogenicity of the mRNA vaccine candidate compared to the inactivated split influenza vaccine and its enhanced ability to generate a cross-specific humoral immune response. These findings highlight the potential mRNA technology in overcoming current limitations of influenza vaccines and hold promise for ensuring greater efficacy in preventing seasonal influenza outbreaks.

Differential effects of heating modes on the immunogenic potential of soy-derived peptides released after in vitro infant digestion.

During production of soy-based infant formula, soy protein undergoes heating processes. This study investigated the differential impact of heating modes on the immunogenic potential of peptides in soy protein digests. Wet or dry heating was applied, followed by in vitro gastrointestinal infant digestion. The released peptides were analyzed by LC-MS/MS. Bioinformatics tools were utilized to predict and identify potential linear B-cell and T-cell epitopes, as well as to explore cross-reactivity with other legumes. Subsequently, the peptide intensities of the same potential epitope across different experimental conditions were compared. As a result, we confirmed the previously observed enhancing effect of wet heating on infant digestion and inhibitory effect of dry heating. A total of 8,546 peptides were detected in the digests, and 6,684 peptides were with a score over 80. Among them, 29 potential T-cell epitopes and 27 potential B-cell epitopes were predicted. Cross-reactivity between soy and other legumes, including peanut, pea, chickpea, lentil, kidney bean, and lupine, was also detected. Overall, heating and digestion time could modulate the potential to trigger peptide-induced immune responses.

BCG activation of trained immunity is associated with induction of cross reactive COVID-19 antibodies in a BCG vaccinated population.

BACKGROUND: Pakistan is endemic to a diverse set of parasitic, mycobacterial and viral diseases. The recognition of BCG Trained Immunity (TI) led us to postulate that the continued presence of BCG-TI may play a protective role, previously reported for both infectious and noninfectious conditions. Most of the previous studies have addressed the issue of BCG-TI in the paediatric populations. This study addressed the key issue of maintenance of BCG-TI in a wider age range (adolescent and adults) to identify the strength and quality of the immune responses. OBJECTIVE: To assess the BCG-induced recall responses in healthy individuals by cytokines secreted from the TI network and its potential role in providing cross-protection against COVID-19 and other viral infections. STUDY DESIGN: In this cross-sectional study, healthy young adults and adolescents (n = 20) were recruited from 16-40 years of age, with no prior history of TB treatment, autoimmune, or chronic inflammatory condition. METHODS: BCG-induced cytokine responses were assessed using prototypic markers for cells of the TI network [macrophages [M1 (TNFα, IFNγ), M2 (IL10)], NK (IL2), Gamma delta (γδ) T (IL17, IL4)] and SARS CoV2 IgG antibodies against RBD using short-term (12 hrs.) cultures assay. RESULTS: Significant differences were observed in the magnitude of recall responses to BCG with macrophage cytokines showing the highest mean levels of TNFα (9148 pg/ml) followed by IL10 (488 pg/ml) and IFNγ (355 pg/ml). The ratio of unstimulated vs.BCG-stimulated cytokines was 132 fold higher for TNFα, 40 fold fo r IL10, and 27 fold for IFNγ. Furthermore, SARS-CoV-2 antibodies were also detected in unstimulated plasma which showed cross reactivity with BCG. CONCLUSION: The presence of cross reactive antibodies to SARS-CoV-2 and the relative ratio of pro- and anti-inflammatory cytokines secreted by activated TI cellular network may play a pivotal role in protection in the early stages of infection as observed during the COVID-19 pandemic in the younger age groups resulting in lower morbidity and mortality.

Cross-Reactive Antibody Responses to Coronaviruses Elicited by SARS-CoV-2 Infection or Vaccination.

BACKGROUND: The newly emerged SARS-CoV-2 possesses shared antigenic epitopes with other human coronaviruses. We investigated if COVID-19 vaccination or SARS-CoV-2 infection may boost cross-reactive antibodies to other human coronaviruses. METHODS: Prevaccination and postvaccination sera from SARS-CoV-2 naïve healthy subjects who received three doses of the mRNA vaccine (BioNTech, BNT) or the inactivated vaccine (CoronaVac, CV) were used to monitor the level of cross-reactive antibodies raised against other human coronaviruses by enzyme-linked immunosorbent assay. In comparison, convalescent sera from COVID-19 patients with or without prior vaccination history were also tested. Pseudoparticle neutralization assay was performed to detect neutralization antibody against MERS-CoV. RESULTS: Among SARS-CoV-2 infection-naïve subjects, BNT or CV significantly increased the anti-S2 antibodies against Betacoronaviruses (OC43 and MERS-CoV) but not Alphacoronaviruses (229E). The prevaccination antibody response to the common cold human coronaviruses did not negatively impact the postvaccination antibody response to SARS-CoV-2. Cross-reactive antibodies that binds to the S2 protein of MERS-CoV were similarly detected from the convalescent sera of COVID-19 patients with or without vaccination history. However, these anti-S2 antibodies do not possess neutralizing activity in MERS-CoV pseudoparticle neutralization tests. CONCLUSIONS: Our results suggest that SARS-CoV-2 infection or vaccination may potentially modulate population immune landscape against previously exposed or novel human coronaviruses. The findings have implications for future sero-epidemiological studies on MERS-CoV.

[Molecular mimicry between human thyroid peroxidase, thyroglobulin, cosinophil peroxidase, IL-24 and microorganisms antigens]. / Mimetismo molecular entre peroxidasa tiroidea humana, tiroglobulina, peroxidasa de eosinófilos, IL-24 y antígenos de microorganismos.

OBJECTIVE: Identify molecular mimicry between TPO, eosinophil peroxidase (EPX), thyroglobulin and IL24 and microorganism antigens. METHODS: Through in silico analysis, we performed local alignments between human and microorganism antigens with PSI-BLAST. Proteins that did not present a 3D structure were modeled by homology through the Swiss Modeller server and epitope prediction was performed through Ellipro. Epitopes were located in the 3D models using PYMOL software. RESULTS: A total of 38 microorganism antigens (parasites, bacteria) had identities between 30% and 45%, being the highest with Anisakis simplex. The alignment between 2 candidate proteins from A. simplex and EPX presented significant values, with identities of 43 and 44%. In bacteria, Campylobacter jejuni presented the highest identity with thyroglobulin (35%). 220 linear and conformational epitopes of microorganism antigens were predicted. Peroxidasin-like proteins from Toxocara canis and Trichinella pseudospiralis presented 10 epitopes similar to TPO and EPX, as possible molecules triggering cross-reactivity. No virus presented identity with the human proteins studied. CONCLUSION: TPO and EPX antigens shared potential cross-reactive epitopes with bacterial and nematode proteins, suggesting that molecular mimicry could be a mechanism that explains the relationship between infections and urticaria/hypothyroidism. In vitro work is needed to demonstrate the results obtained in the in silico analysis.

OBJETIVO: Identificar mimetismo molecular entre TPO, eosinofil peroxidasa (EPX), tiroglobulina e IL24 y antígenos de microorganismos. MÉTODOS: A través de análisis in silico, realizamos los alineamientos locales entre los antígenos humanos y de microorganismos con PSI-BLAST. Las proteínas que no presentaban estructura 3D, fueron modeladas por homología a través del servidor Swiss Modeller y se realizó una predicción de epítopes a través de Ellipro. Los epítopes se localizaron en los modelos 3D utilizando el software PYMOL. RESULTADOS: Un total de 38 antígenos de microorganismos (parásitos y bacterias), tuvieron identidades entre 30 y 45%, siendo los más altos con Anisakis simplex. El alineamiento entre dos proteínas candidatas de A. simplex y EPX presentaron valores importantes, con identidades de 43 y 44%. En las bacterias, Campylobacter jejuni presentó la mayor identidad con tiroglobulina (35%). Se predijeron 220 epítopes lineales y conformacionales de antígenos de microorganismos. Las proteínas similares a la peroxidasina de Toxocara canis y Trichinella pseudospiralis presentaron diez epítopes similares a TPO y EPX, como posibles moléculas desencadenantes de una reactividad cruzada. Ningún virus presentó identidad con las proteínas humanas estudiadas. CONCLUSIÓN: Los antígenos TPO y EPX compartieron potenciales epítopes de reacción cruzada con proteínas bacterianas y nematodos, lo que sugiere que el mimetismo molecular podría ser un mecanismo que explique la relación entre infecciones y la urticaria/hipotiroidismo. Se necesitan trabajos in vitro que demuestren los resultados obtenidos en el análisis in silico.

Japanese encephalitis virus E protein domain III immunization mediates cross-protection against Zika virus in mice via antibodies and CD8+T cells.

Zika virus (ZIKV) and Japanese encephalitis virus (JEV) are antigenically related flaviviruses that co-circulate in many countries/territories. The interaction between the two viruses needs to be determined. Recent findings by ourselves and other labs showed that JEV-elicited antibodies (Abs) and CD8+T cells exacerbate and protect against subsequent ZIKV infection, respectively. However, the impact of JEV envelope (E) protein domain III (EDIII)-induced immune responses on ZIKV infection is unclear. We show here that sera from JEV-EDIII-vaccinated mice cross-react with ZIKV-EDIII in vitro, and transfer of the same sera to mice significantly decreases death upon lethal ZIKV infection at a dose-dependent manner. Maternally acquired anti-JEV-EDIII Abs also significantly reduce the mortality of neonatal mice born to JEV-EDIII-immune mothers post ZIKV challenge. Similarly, transfer of ZIKV-EDIII-reactive IgG purified from JEV-vaccinated humans increases the survival of ZIKV-infected mice. Notably, transfer of an extremely low volume of JEV-EDIII-immune sera or ZIKV-EDIII-reactive IgG does not mediate the Ab-mediated enhancement (ADE) of ZIKV infection. Similarly, transfer of JEV-EDIII-elicited CD8+T cells protects recipient mice against ZIKV challenge. These results demonstrate that JEV-EDIII-induced immune components including Abs and T cells have protective roles in ZIKV infection, suggesting EDIII is a promising immunogen for developing effective and safety JEV vaccine.

Antibodies Induced by Smallpox Vaccination after at Least 45 Years Cross-React with and In Vitro Neutralize Mpox Virus: A Role for Polyclonal B Cell Activation?

AIMS: To evaluate whether antibodies specific for the vaccinia virus (VV) are still detectable after at least 45 years from immunization. To confirm that VV-specific antibodies are endowed with the capacity to neutralize Mpox virus (MPXV) in vitro. To test a possible role of polyclonal non-specific activation in the maintenance of immunologic memory. METHODS: Sera were collected from the following groups: smallpox-vaccinated individuals with or without latent tuberculosis infection (LTBI), unvaccinated donors, and convalescent individuals after MPXV infection. Supernatant of VV- or MPXV-infected Vero cells were inactivated and used as antigens in ELISA or in Western blot (WB) analyses. An MPXV plaque reduction neutralization test (PRNT) was optimized and performed on study samples. VV- and PPD-specific memory T cells were measured by flow cytometry. RESULTS: None of the smallpox unvaccinated donors tested positive in ELISA or WB analysis and their sera were unable to neutralize MPXV in vitro. Sera from all the individuals convalescing from an MPXV infection tested positive for anti-VV or MPXV IgG with high titers and showed MPXV in vitro neutralization capacity. Sera from most of the vaccinated individuals showed IgG anti-VV and anti-MPXV at high titers. WB analyses showed that positive sera from vaccinated or convalescent individuals recognized both VV and MPXV antigens. Higher VV-specific IgG titer and specific T cells were observed in LTBI individuals. CONCLUSIONS: ELISA and WB performed using supernatant of VV- or MPXV-infected cells are suitable to identify individuals vaccinated against smallpox at more than 45 years from immunization and individuals convalescing from a recent MPXV infection. ELISA and WB results show a good correlation with PRNT. Data confirm that a smallpox vaccination induces a long-lasting memory in terms of specific IgG and that antibodies raised against VV may neutralize MPXV in vitro. Finally, higher titers of VV-specific antibodies and higher frequency of VV-specific memory T cells in LTBI individuals suggest a role of polyclonal non-specific activation in the maintenance of immunologic memory.

Development of Paper-Based Fluorescent Molecularly Imprinted Polymer Sensor for Rapid Detection of Lumpy Skin Disease Virus.

Lumpy Skin Disease (LSD) is a notifiable viral disease caused by Lumpy Skin Disease virus (LSDV). It is usually associated with high economic losses, including a loss of productivity, infertility, and death. LSDV shares genetic and antigenic similarities with Sheep pox virus (SPV) and Goat pox (GPV) virus. Hence, the LSDV traditional diagnostic tools faced many limitations regarding sensitivity, specificity, and cross-reactivity. Herein, we fabricated a paper-based turn-on fluorescent Molecularly Imprinted Polymer (MIP) sensor for the rapid detection of LSDV. The LSDV-MIPs sensor showed strong fluorescent intensity signal enhancement in response to the presence of the virus within minutes. Our sensor showed a limit of detection of 101 log10 TCID50/mL. Moreover, it showed significantly higher specificity to LSDV relative to other viruses, especially SPV. To our knowledge, this is the first record of a paper-based rapid detection test for LSDV depending on fluorescent turn-on behavior.

Computational Characterization of Membrane Proteins as Anticancer Targets: Current Challenges and Opportunities.

Cancer remains a leading cause of mortality worldwide and calls for novel therapeutic targets. Membrane proteins are key players in various cancer types but present unique challenges compared to soluble proteins. The advent of computational drug discovery tools offers a promising approach to address these challenges, allowing for the prioritization of "wet-lab" experiments. In this review, we explore the applications of computational approaches in membrane protein oncological characterization, particularly focusing on three prominent membrane protein families: receptor tyrosine kinases (RTKs), G protein-coupled receptors (GPCRs), and solute carrier proteins (SLCs). We chose these families due to their varying levels of understanding and research data availability, which leads to distinct challenges and opportunities for computational analysis. We discuss the utilization of multi-omics data, machine learning, and structure-based methods to investigate aberrant protein functionalities associated with cancer progression within each family. Moreover, we highlight the importance of considering the broader cellular context and, in particular, cross-talk between proteins. Despite existing challenges, computational tools hold promise in dissecting membrane protein dysregulation in cancer. With advancing computational capabilities and data resources, these tools are poised to play a pivotal role in identifying and prioritizing membrane proteins as personalized anticancer targets.