Enhancing Nanobody Immunoassays through Ferritin Fusion: Construction of a Salmonella-Specific Fenobody for Improved Avidity and Sensitivity.

Nanobodies (Nbs) serve as powerful tools in immunoassays. However, their small size and monovalent properties pose challenges for practical application. Multimerization emerges as a significant strategy to address these limitations, enhancing the utilization of nanobodies in immunoassays. Herein, we report the construction of a Salmonella-specific fenobody (Fb) through the fusion of a nanobody to ferritin, resulting in a self-assembled 24-valent nanocage-like structure. The fenobody exhibits a 35-fold increase in avidity compared to the conventional nanobody while retaining good thermostability and specificity. Leveraging this advancement, three ELISA modes were designed using Fb as the capture antibody, along with unmodified Nb422 (FbNb-ELISA), biotinylated Nb422 (FbBio-ELISA), and phage-displayed Nb422 (FbP-ELISA) as the detection antibody, respectively. Notably, the FbNb-ELISA demonstrates a detection limit (LOD) of 3.56 × 104 CFU/mL, which is 16-fold lower than that of FbBio-ELISA and similar to FbP-ELISA. Moreover, a fenobody and nanobody sandwich chemiluminescent enzyme immunoassay (FbNb-CLISA) was developed by replacing the TMB chromogenic substrate with luminal, resulting in a 12-fold reduction in the LOD. Overall, the ferritin-displayed technology represents a promising methodology for enhancing the detection performance of nanobody-based sandwich ELISAs, thereby expanding the applicability of Nbs in food detection and other fields requiring multivalent modification.

A ferritin-based nanoparticle displaying a neutralizing epitope for foot-and-mouth disease virus (FMDV) confers partial protection in guinea pigs.

BACKGROUND: Foot-and-mouth disease (FMD) is a devastating disease affecting cloven-hoofed animals, that leads to significant economic losses in affected countries and regions. Currently, there is an evident inclination towards the utilization of nanoparticles as powerful platforms for innovative vaccine development. Therefore, this study developed a ferritin-based nanoparticle (FNP) vaccine that displays a neutralizing epitope of foot-and-mouth disease virus (FMDV) VP1 (aa 140-158) on the surface of FNP, and evaluated the immunogenicity and protective efficacy of these FNPs in mouse and guinea pig models to provide a strategy for developing potential FMD vaccines. RESULTS: This study expressed the recombinant proteins Hpf, HPF-NE and HPF-T34E via an E. coli expression system. The results showed that the recombinant proteins Hpf, Hpf-NE and Hpf-T34E could be effectively assembled into nanoparticles. Subsequently, we evaluated the immunogenicity of the Hpf, Hpf-NE and Hpf-T34E proteins in mice, as well as the immunogenicity and protectiveness of the Hpf-T34E protein in guinea pigs. The results of the mouse experiment showed that the immune efficacy in the Hpf-T34E group was greater than the Hpf-NE group. The results from guinea pigs immunized with Hpf-T34E showed that the immune efficacy was largely consistent with the immunogenicity of the FMD inactivated vaccine (IV) and could confer partial protection against FMDV challenge in guinea pigs. CONCLUSIONS: The Hpf-T34E nanoparticles stand out as a superior choice for a subunit vaccine candidate against FMD, offering effective protection in FMDV-infected model animals. FNP-based vaccines exhibit excellent safety and immunogenicity, thus representing a promising strategy for the continued development of highly efficient and safe FMD vaccines.

Development of a Ferritin Protein Nanoparticle Vaccine with PRRSV GP5 Protein.

Porcine reproductive and respiratory syndrome virus (PRRSV) presents a significant threat to the global swine industry. The development of highly effective subunit nanovaccines is a promising strategy for preventing PRRSV variant infections. In this study, two different types of ferritin (Ft) nanovaccines targeting the major glycoprotein GP5, named GP5m-Ft and (Bp-IVp)3-Ft, were constructed and evaluated as vaccine candidates for PRRSV. Transmission electron microscopy (TEM) and dynamic light scattering (DLS) demonstrated that both purified GP5m-Ft and (Bp-IVp)3-Ft proteins could self-assemble into nanospheres. A comparison of the immunogenicity of GP5m-Ft and (Bp-IVp)3-Ft with an inactivated PRRSV vaccine in BALB/c mice revealed that mice immunized with GP5m-Ft exhibited the highest ELISA antibody levels, neutralizing antibody titers, the lymphocyte proliferation index, and IFN-γ levels. Furthermore, vaccination with the GP5m-Ft nanoparticle effectively protected piglets against a highly pathogenic PRRSV challenge. These findings suggest that GP5m-Ft is a promising vaccine candidate for controlling PRRS.

A ferritin nanoparticle vaccine based on the hemagglutinin extracellular domain of swine influenza A (H1N1) virus elicits protective immune responses in mice and pigs.

Introduction: Swine influenza viruses (SIVs) pose significant economic losses to the pig industry and are a burden on global public health systems. The increasing complexity of the distribution and evolution of different serotypes of influenza strains in swine herds escalates the potential for the emergence of novel pandemic viruses, so it is essential to develop new vaccines based on swine influenza. Methods: Here, we constructed a self-assembling ferritin nanoparticle vaccine based on the hemagglutinin (HA) extracellular domain of swine influenza A (H1N1) virus using insect baculovirus expression vector system (IBEVS), and after two immunizations, the immunogenicities and protective efficacies of the HA-Ferritin nanoparticle vaccine against the swine influenza virus H1N1 strain in mice and piglets were evaluated. Results: Our results demonstrated that HA-Ferritin nanoparticle vaccine induced more efficient immunity than traditional swine influenza vaccines. Vaccination with the HA-Ferritin nanoparticle vaccine elicited robust hemagglutinin inhibition titers and antigen-specific IgG antibodies and increased cytokine levels in serum. MF59 adjuvant can significantly promote the humoral immunity of HA-Ferritin nanoparticle vaccine. Furthermore, challenge tests showed that HA-Ferritin nanoparticle vaccine conferred full protection against lethal challenge with H1N1 virus and significantly decreased the severity of virus-associated lung lesions after challenge in both BALB/c mice and piglets. Conclusion: Taken together, these results indicate that the hemagglutinin extracellular-based ferritin nanoparticle vaccine may be a promising vaccine candidate against SIVs infection.

Immunogenicity and safety of a self-assembling ZIKV nanoparticle vaccine in mice.

Zika virus (ZIKV) is a mosquito-borne flavivirus that highly susceptibly causes Guillain-Barré syndrome and microcephaly in newborns. Vaccination is one of the most effective measures for preventing infectious diseases. However, there is currently no approved vaccine to prevent ZIKV infection. Here, we developed nanoparticle (NP) vaccines by covalently conjugating self-assembled 24-subunit ferritin to the envelope structural protein subunit of ZIKV to achieve antigen polyaggregation. The immunogenicityof the NP vaccine was evaluated in mice. Compared to monomer vaccines, the NP vaccine achieved effective antigen presentation, promoted the differentiation of follicular T helper cells in lymph nodes, and induced significantly greater antigen-specific humoral and cellular immune responses. Moreover, the NP vaccine enhanced high-affinity antigen-specific IgG antibody levels, increased secretion of the cytokines IL-4 and IFN-γ by splenocytes, significantly activated T/B lymphocytes, and improved the generation of memory T/B cells. In addition, no significant adverse reactions occurred when NP vaccine was combined with adjuvants. Overall, ferritin-based NP vaccines are safe and effective ZIKV vaccine candidates.

A highly effective ferritin-based divalent nanoparticle vaccine shields Syrian hamsters against lethal Nipah virus.

The Nipah virus (NiV), a highly deadly bat-borne paramyxovirus, poses a substantial threat due to recurrent outbreaks in specific regions, causing severe respiratory and neurological diseases with high morbidity. Two distinct strains, NiV-Malaysia (NiV-M) and NiV-Bangladesh (NiV-B), contribute to outbreaks in different geographical areas. Currently, there are no commercially licensed vaccines or drugs available for prevention or treatment. In response to this urgent need for protection against NiV and related henipaviruses infections, we developed a novel homotypic virus-like nanoparticle (VLP) vaccine co-displaying NiV attachment glycoproteins (G) from both strains, utilizing the self-assembling properties of ferritin protein. In comparison to the NiV G subunit vaccine, our nanoparticle vaccine elicited significantly higher levels of neutralizing antibodies and provided complete protection against a lethal challenge with NiV infection in Syrian hamsters. Remarkably, the nanoparticle vaccine stimulated the production of antibodies that exhibited superior cross-reactivity to homologous or heterologous henipavirus. These findings underscore the potential utility of ferritin-based nanoparticle vaccines in providing both broad-spectrum and long-term protection against NiV and emerging zoonotic henipaviruses challenges.

Construction and immune effect evaluation of the S protein heptad repeat-based nanoparticle vaccine against porcine epidemic diarrhea virus.

Porcine epidemic diarrhea virus (PEDV), a highly virulent enteropathogenic coronavirus, is a significant threat to the pig industry. High frequency mutations in the PEDV genome have limited the effectiveness of current vaccines in providing immune protection. Developing efficient vaccines that can quickly adapt to mutant strains is a challenging but crucial task. In this study, we chose the pivotal protein heptad repeat (HR) responsible for coronavirus entry into host cells, as the vaccine antigen. HR-Fer nanoparticles prepared using ferritin were evaluated them as PEDV vaccine candidates. Nanoparticle vaccines elicited stronger neutralizing antibody responses in mice compared to monomer vaccines. Additionally, HR protein delivered via nanoparticles increased antigen uptake by antigen-presenting cells in vitro by 2.75-fold. The collective results suggest that HR can be used as antigens for vaccines, and the HR vaccine based on ferritin nanoparticles significantly enhances immunogenicity.

Enhancing Tumor Immunotherapy by Multivalent Anti-PD-L1 Nanobody Assembled via Ferritin Nanocage.

Increasing immunotherapy response rate and durability can lead to significant improvements in cancer care. To address this challenge, a novel multivalent immune checkpoint therapeutic platform is constructed through site-specific ligation of anti-PD-L1 nanobody (Nb) on ferritin (Ftn) nanocage. Nb-Ftn blocks PD-1/PD-L1 interaction and downregulates PD-L1 levels via endocytosis-induced degradation. In addition, the cage structure of Ftn allows encapsulation of indocyanine green (ICG), an FDA-approved dye. Photothermal treatment with Nb-Ftn@ICG induces immunogenic death of tumor cells, which improves systemic immune response via maturation of dendritic cells and enhanced infiltration of T cells. Moreover, Nb-Ftn encapsulation significantly enhances cellular uptake, tumor accumulation and retention of ICG. In vivo assays showed that this nanoplatform ablates the primary tumor, suppresses abscopal tumors and inhibits tumor metastasis, leading to a prolonged survival rate. This work presents a novel strategy for improving cancer immunotherapy using multivalent nanobody-ferritin conjugates as immunological targeting and enhancing carriers.

Surface Engineering of Graphene through Heterobifunctional Supramolecular-Covalent Scaffolds for Rapid COVID-19 Biomarker Detection.

Graphene is a two-dimensional semiconducting material whose application for diagnostics has been a real game-changer in terms of sensitivity and response time, variables of paramount importance to stop the COVID-19 spreading. Nevertheless, strategies for the modification of docking recognition and antifouling elements to obtain covalent-like stability without the disruption of the graphene band structure are still needed. In this work, we conducted surface engineering of graphene through heterofunctional supramolecular-covalent scaffolds based on vinylsulfonated-polyamines (PA-VS). In these scaffolds, one side binds graphene through multivalent π-π interactions with pyrene groups, and the other side presents vinylsulfonated pending groups that can be used for covalent binding. The construction of PA-VS scaffolds was demonstrated by spectroscopic ellipsometry, Raman spectroscopy, and contact angle measurements. The covalent binding of -SH, -NH2, or -OH groups was confirmed, and it evidenced great chemical versatility. After field-effect studies, we found that the PA-VS-based scaffolds do not disrupt the semiconducting properties of graphene. Moreover, the scaffolds were covalently modified with poly(ethylene glycol) (PEG), which improved the resistance to nonspecific proteins by almost 7-fold compared to the widely used PEG-monopyrene approach. The attachment of recognition elements to PA-VS was optimized for concanavalin A (ConA), a model lectin with a high affinity to glycans. Lastly, the platform was implemented for the rapid, sensitive, and regenerable recognition of SARS-CoV-2 spike protein and human ferritin in lab-made samples. Those two are the target molecules of major importance for the rapid detection and monitoring of COVID-19-positive patients. For that purpose, monoclonal antibodies (mAbs) were bound to the scaffolds, resulting in a surface coverage of 436 ± 30 ng/cm2. KD affinity constants of 48.4 and 2.54 nM were obtained by surface plasmon resonance (SPR) spectroscopy for SARS-CoV-2 spike protein and human ferritin binding on these supramolecular scaffolds, respectively.

Cell Population Data and Serum Polyclonal Immunoglobulin Free Light Chains in the Assessment of COVID-19 Severity.

Distinguishing between severe and nonsevere COVID-19 to ensure adequate healthcare quality and efficiency is a challenge for the healthcare system. The aim of this study was to assess the usefulness of CBC parameters together with analysis of FLC serum concentration in risk stratification of COVID-19. MATERIALS AND METHODS: CBC was analyzed in 735 COVID ICU, COVID non-ICU, and non-COVID ICU cases. FLC concentration was analyzed in 133 of them. RESULTS: COVID ICU had neutrophils and lymphocytes with the greatest size, granularity, and nucleic acid content. Significant differences in concentrations of κ and λ FLCs were shown between COVID ICU and COVID non-ICU. However, no difference was found in the κ/λ ratio between these groups, and the ratio stayed within the reference value, which indicates the presence of polyclonal FLCs. FLC κ measurement has significant power to distinguish between severe COVID-19 and nonsevere COVID-19 (AUC = 0.7669), with a sensitivity of 86.67% and specificity of 93.33%. The κ coefficients' odds ratio of 3.0401 was estimated. CONCLUSION: It can be concluded that the results obtained from the measure of free light immunoglobulin concentration in serum are useful in distinguishing between severe and nonsevere COVID-19.

Corona Virus Disease-19 serology, inflammatory markers, hospitalizations, case finding, and aging.

Most deaths from severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection occur in older subjects. We assessed the utility of serum inflammatory markers interleukin-6 (IL-6), C reactive protein (CRP), and ferritin (Roche, Indianapolis, IN), and SARS-CoV-2 immunoglobulin G (IgG), immunoglobulin M (IgM), and neutralizing antibodies (Diazyme, Poway, CA). In controls, non-hospitalized subjects, and hospitalized subjects assessed for SARS-CoV-2 RNA (n = 278), median IgG levels in arbitrary units (AU)/mL were 0.05 in negative subjects, 14.83 in positive outpatients, and 30.61 in positive hospitalized patients (P<0.0001). Neutralizing antibody levels correlated significantly with IgG (r = 0.875; P<0.0001). Having combined values of IL-6 ≥10 pg/mL and CRP ≥10 mg/L occurred in 97.7% of inpatients versus 1.8% of outpatients (odds ratio 3,861, C statistic 0.976, P = 1.00 x 10-12). Antibody or ferritin levels did not add significantly to predicting hospitalization. Antibody testing in family members and contacts of SARS-CoV-2 RNA positive cases (n = 759) was invaluable for case finding. Persistent IgM levels were associated with chronic COVID-19 symptoms. In 81,624 screened subjects, IgG levels were positive (≥1.0 AU/mL) in 5.21%, while IgM levels were positive in 2.96% of subjects. In positive subjects median IgG levels in AU/mL were 3.14 if <30 years of age, 4.38 if 30-44 years of age, 7.89 if 45-54 years of age, 9.52 if 55-64 years of age, and 10.64 if ≥65 years of age (P = 2.96 x 10-38). Our data indicate that: 1) combined IL-6 ≥10 pg/mL and CRP ≥10 mg/L identify SARS-CoV-2 positive subjects requiring hospitalization; 2) IgG levels were significantly correlated with neutralizing antibody levels with a wide range of responses; 3) IgG levels have significant utility for case finding in exposed subjects; 4) persistently elevated IgM levels are associated with chronic symptoms; and 5) IgG levels are significantly higher in positive older subjects than their younger counterparts.

A novel ferritin subunit gene from Asian green mussel, Perna viridis (Linnaeus, 1758).

Iron sequestration through ferritin forms a major part of innate immune response in molluscs and detailed understanding of ferritin gene and its functions can be directly applied in infection and disease management studies. Accordingly, identification and detailed molecular characterization of a ferritin subunit gene from a commercially significant marine mussel Perna viridis was targeted. Molecular screening using degenerate primers in total mantle RNA resulted in the amplification of a novel ferritin gene fragment having <87% identity to the reported ferritin gene sequences. Rapid amplification of cDNA ends-PCR was followed to generate complete cDNA sequence of P.viridis ferritin (PvFer). The complete cDNA was found to be 798 bp, containing an open reading frame of 522 bp, 5' untranslated region (UTR) of 112 bp and 3' UTR of 165 bp. The 5' UTR and 3' UTR were shown to contain an iron response element (IRE) and a polyadenylation signal (767AATAAA772) with poly (A) tail, respectively. Prediction of stem loop structure revealed that, PvFer-IRE can be folded into a typical secondary stem loop structure, having 5-CAGUGA-3' loop, proximal stem of five paired bases followed by a bulged cysteine, and six nucleotide bottom stem, indicating that expression of PvFer is regulated by iron at the translational level. ORF was found to encode 175 amino acid protein with calculated molecular mass of 19.97 kDa and isoelectric point of 4.97. Examination for signal peptide and phylogenetic analysis confirmed that PvFer belonged to cytosolic ferritins of molluscs. Conserved domain analysis showed that PvFer contained both ferroxidase diiron center and ferrihydrite nucleation center, analogous to ferritin M subunit of bony fishes and amphibians. However, amino acid sequence and glycosylation site showed more homology to vertebrate ferritin H subunits. Predicted 3D models of PvFer resembled the typical spatial features of ferritin proteins. The study forms the first comprehensive identification of a ferritin subunit gene in a true/common mussel (Order: Mytilida). Further, the detailed molecular phylogeny conducted through the present study revealed certain thought provoking insights on ferritin genes of the phylum Mollusca.

Broad neutralization of H1 and H3 viruses by adjuvanted influenza HA stem vaccines in nonhuman primates.

Seasonal influenza vaccines confer protection against specific viral strains but have restricted breadth that limits their protective efficacy. The H1 and H3 subtypes of influenza A virus cause most of the seasonal epidemics observed in humans and are the major drivers of influenza A virus-associated mortality. The consequences of pandemic spread of COVID-19 underscore the public health importance of prospective vaccine development. Here, we show that headless hemagglutinin (HA) stabilized-stem immunogens presented on ferritin nanoparticles elicit broadly neutralizing antibody (bnAb) responses to diverse H1 and H3 viruses in nonhuman primates (NHPs) when delivered with a squalene-based oil-in-water emulsion adjuvant, AF03. The neutralization potency and breadth of antibodies isolated from NHPs were comparable to human bnAbs and extended to mismatched heterosubtypic influenza viruses. Although NHPs lack the immunoglobulin germline VH1-69 residues associated with the most prevalent human stem-directed bnAbs, other gene families compensated to generate bnAbs. Isolation and structural analyses of vaccine-induced bnAbs revealed extensive interaction with the fusion peptide on the HA stem, which is essential for viral entry. Antibodies elicited by these headless HA stabilized-stem vaccines neutralized diverse H1 and H3 influenza viruses and shared a mode of recognition analogous to human bnAbs, suggesting that these vaccines have the potential to confer broadly protective immunity against diverse viruses responsible for seasonal and pandemic influenza infections in humans.

The Versatile Manipulations of Self-Assembled Proteins in Vaccine Design.

Protein assemblies provide unique structural features which make them useful as carrier molecules in biomedical and chemical science. Protein assemblies can accommodate a variety of organic, inorganic and biological molecules such as small proteins and peptides and have been used in development of subunit vaccines via display parts of viral pathogens or antigens. Such subunit vaccines are much safer than traditional vaccines based on inactivated pathogens which are more likely to produce side-effects. Therefore, to tackle a pandemic and rapidly produce safer and more effective subunit vaccines based on protein assemblies, it is necessary to understand the basic structural features which drive protein self-assembly and functionalization of portions of pathogens. This review highlights recent developments and future perspectives in production of non-viral protein assemblies with essential structural features of subunit vaccines.

Weathering the COVID-19 storm: Lessons from hematologic cytokine syndromes.

A subset of patients with severe COVID-19 develop profound inflammation and multi-organ dysfunction consistent with a "Cytokine Storm Syndrome" (CSS). In this review we compare the clinical features, diagnosis, and pathogenesis of COVID-CSS with other hematological CSS, namely secondary hemophagocytic lymphohistiocytosis (sHLH), idiopathic multicentric Castleman disease (iMCD), and CAR-T cell therapy associated Cytokine Release Syndrome (CRS). Novel therapeutics targeting cytokines or inhibiting cell signaling pathways have now become the mainstay of treatment in these CSS. We review the evidence for cytokine blockade and attenuation in these known CSS as well as the emerging literature and clinical trials pertaining to COVID-CSS. Established markers of inflammation as well as cytokine levels are compared and contrasted between these four entities in order to establish a foundation for future diagnostic criteria of COVID-CSS.

An immune-based biomarker signature is associated with mortality in COVID-19 patients.

Immune and inflammatory responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) contribute to disease severity of coronavirus disease 2019 (COVID-19). However, the utility of specific immune-based biomarkers to predict clinical outcome remains elusive. Here, we analyzed levels of 66 soluble biomarkers in 175 Italian patients with COVID-19 ranging from mild/moderate to critical severity and assessed type I IFN-, type II IFN-, and NF-κB-dependent whole-blood transcriptional signatures. A broad inflammatory signature was observed, implicating activation of various immune and nonhematopoietic cell subsets. Discordance between IFN-α2a protein and IFNA2 transcript levels in blood suggests that type I IFNs during COVID-19 may be primarily produced by tissue-resident cells. Multivariable analysis of patients' first samples revealed 12 biomarkers (CCL2, IL-15, soluble ST2 [sST2], NGAL, sTNFRSF1A, ferritin, IL-6, S100A9, MMP-9, IL-2, sVEGFR1, IL-10) that when increased were independently associated with mortality. Multivariate analyses of longitudinal biomarker trajectories identified 8 of the aforementioned biomarkers (IL-15, IL-2, NGAL, CCL2, MMP-9, sTNFRSF1A, sST2, IL-10) and 2 additional biomarkers (lactoferrin, CXCL9) that were substantially associated with mortality when increased, while IL-1α was associated with mortality when decreased. Among these, sST2, sTNFRSF1A, IL-10, and IL-15 were consistently higher throughout the hospitalization in patients who died versus those who recovered, suggesting that these biomarkers may provide an early warning of eventual disease outcome.

Immunization with a recombinant fusion of porcine reproductive and respiratory syndrome virus modified GP5 and ferritin elicits enhanced protective immunity in pigs.

Porcine reproductive and respiratory syndrome (PRRS) has caused huge economic losses in the swine industry worldwide. Live and inactivated vaccines have only been partially successful in generating protective immune responses. The PRRS virus (PRRSV) glycoprotein 5 (GP5) is a major viral antigenic target and is thus suitable for development of genetically engineered PRRSV vaccines. Here, a modified GP5 and ferritin were fused and expressed using a baculovirus system to generate a GP5m-ferritin nanoparticle vaccine. We demonstrated that the GP5m-ferritin vaccine elicited higher serum antibody titers in pigs than inactivated PRRSV. Moreover, immunization with GP5m-Ft promoted a Th1-dominant cellular immune response and enhanced specific T-lymphocyte immune responses. GP5m-ferritin-vaccinated pigs had significantly lower mean rectal temperatures, respiratory scores, viremia, and macroscopic and microscopic lung lesion scores post-challenge compared with unvaccinated pigs. These results indicated that GP5m-ferritin subunit vaccines can elicit specific protective immune responses and represent promising vaccine candidates.

IL-15 is a biomarker involved in the development of rapidly progressive interstitial lung disease complicated with polymyositis/dermatomyositis.

BACKGROUND: Polymyositis/dermatomyositis (PM/DM) is an autoimmune disease that is sometimes complicated with rapidly progressive interstitial lung disease (RPILD). However, serum and lung biomarkers that can predict RPILD development remain unclear. OBJECTIVES: To determine potential serum and lung biomarkers that can predict RPILD development in patients with PM/DM-ILD. METHODS: In total, 49 patients with PM/DM-ILD were enrolled. We measured the serum levels of 41 cytokines/chemokines, ferritin and anti-MDA5 antibody, compared them between the RPILD (n = 23) and non-RPILD (n = 26) groups, and ranked them by their importance through random forest analysis. To distinguish the two groups, we determined biomarker combinations by logistic regression analysis. We also measured the bronchoalveolar lavage fluid (BALF) levels of 41 cytokines/chemokines. Using immunohistochemistry, we examined IL-15 expression in lung tissues. The IL-15 production was also investigated using A549 and BEAS-2B cells. RESULTS: The RPILD group had significantly higher IL-15, IL-1RA, IL-6, CXCL10, VCAM-1, anti-MDA5 antibody and ferritin serum levels than the non-RPILD group, but it had a significantly low CCL22 level. Meanwhile, anti-MDA5 antibody, IL-15, CXCL8, CCL22, IL-1RA and ferritin were the best combination to distinguish the two groups. IL-15 and CCL22 were also predictive marker for RPILD development in anti-MDA5 antibody-positive patients. Additionally, the RPILD group had significantly high IL-15 levels in BALF. The lung tissues expressed IL-15, which increased after cytokine stimulation in the A549 cells. CONCLUSION: This study identified a combination of biomarkers predicting PM/DM-RPILD progression, and IL-15 is an important cytokine for predicting RPILD development and reflecting ILD severity.

Broad Neutralization Responses Against Oncogenic Human Papillomaviruses Induced by a Minor Capsid L2 Polytope Genetically Incorporated Into Bacterial Ferritin Nanoparticles.

Cervical cancer remains a global health burden despite the introduction of highly effective vaccines for the prophylaxis of causative human papillomavirus infection (HPV). Current efforts to eradicate cervical cancer focus on the development of broadly protective, cost-effective approaches. HPV minor capsid protein L2 is being recognized as a promising alternative to the major capsid protein L1 because of its ability to induce responses against a wider range of different HPV types. However, a major limitation of L2 as a source of cross-neutralizing epitopes is its lower immunogenicity compared to L1 when assembled into VLPs. Various approaches have been proposed to overcome this limitation, we developed and tested ferritin-based bio-nanoparticles displaying tandemly repeated L2 epitopes from eight different HPV types grafted onto the surface of Pyrococcus furiosus thioredoxin (Pf Trx). Genetic fusion of the Pf Trx-L2(8x) module to P. furiosus ferritin (Pf Fe) did not interfere with ferritin self-assembly into an octahedral structure composed by 24 protomers. In guinea pigs and mice, the ferritin super-scaffolded, L2 antigen induced a broadly neutralizing antibody response covering 14 oncogenic and two non-oncogenic HPV types. Immune-responsiveness lasted for at least one year and the resulting antibodies also conferred protection in a cervico-vaginal mouse model of HPV infection. Given the broad organism distribution of thioredoxin and ferritin, we also verified the lack of cross-reactivity of the antibodies elicited against the scaffolds with human thioredoxin or ferritin. Altogether, the results of this study point to P. furiosus ferritin nanoparticles as a robust platform for the construction of peptide-epitope-based HPV vaccines.

Nanoparticle Vaccines Based on the Receptor Binding Domain (RBD) and Heptad Repeat (HR) of SARS-CoV-2 Elicit Robust Protective Immune Responses.

Various vaccine strategies have been proposed in response to the global COVID-19 pandemic, each with unique strategies for eliciting immune responses. Here, we developed nanoparticle vaccines by covalently conjugating the self-assembled 24-mer ferritin to the receptor binding domain (RBD) and/or heptad repeat (HR) subunits of the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) spike (S) protein. Compared to monomer vaccines, nanoparticle vaccines elicited more robust neutralizing antibodies and cellular immune responses. RBD and RBD-HR nanoparticle vaccinated hACE2 transgenic mice vaccinated with RBD and/or RBD-HR nanoparticles exhibited reduced viral load in the lungs after SARS-CoV-2 challenge. RBD-HR nanoparticle vaccines also promoted neutralizing antibodies and cellular immune responses against other coronaviruses. The nanoparticle vaccination of rhesus macaques induced neutralizing antibodies, and T and B cell responses prior to boost immunization; these responses persisted for more than three months. RBD- and HR-based nanoparticles thus present a promising vaccination approach against SARS-CoV-2 and other coronaviruses.