Levels of Natural Antibodies Before and After Immunoglobulin Replacement Treatment Affect the Clinical Phenotype in Common Variable Immunodeficiency.

Natural antibodies (NAbs) occurring in individuals without prior exposure to specific antigens, provide direct first barrier protection against pathogens, and exert immunoregulation thus actively contributing to the maintenance of immune homeostasis, controlling inflammatory processes and preventing autoimmunity. Common variable immunodeficiency (CVID) is a heterogeneous group of disorders characterized by a compromised immune function that brings into focus the role of NAbs. Our aim was to explore whether NAb levels could serve as potential key indicators in CVID for monitoring disease progression and predicting outcomes. In this study, we analyzed a Hellenic cohort of 56 patients with CVID (31 newly diagnosed and 25 under immunoglobulin replacement therapy-IgRT) and 33 healthy controls, for total Ig levels and serum IgM and IgG NAb levels against five informative target-antigens of NAbs, namely, actin, DNA, carbonic anhydrase, F(ab΄)2 fragments of human IgG and TriNitroPhenyl. In addition, follow-up pre- and post- IgRT samples were analyzed in ten (10) patients of our cohort. Results showed that Ig-treated patients exhibited significantly lower IgM NAb levels than untreated patients and healthy controls against all panel antigens. In the follow-up samples, pre-treatment IgM NAb levels negatively correlated with total serum IgM. This imbalance was only partially restored after IgRT, with a significant decrease in IgM NAb levels observed in nine out of ten patients. Moreover, post-treatment patients with recurrent infections presented significantly lower IgM NAb levels, a reduction also observed in patients with bronchiectasis independently of treatment status. On the contrary, post-treatment patients with enteropathy had significantly higher IgM NAb levels against all panel antigens, an increase also noted in patients with autoimmune diseases. Regarding IgG NAbs, replacement therapy restored levels to those of healthy controls. In conclusion, impaired NAb levels are found in CVID patients, particularly related to certain phenotypes. Moreover, the significant decrease in IgM NAb levels after IgRT suggests a potential association with disease course and complications. The results suggest that administration of human IgM NAbs may be an effective combinatorial treatment in selected patients. Further research is needed to understand the functional roles of NAbs in CVID and its complex clinical phenotypes.

A chimeric porcine reproductive and respiratory syndrome virus 1 strain containing synthetic ORF2-6 genes can trigger T follicular helper cell and heterologous neutralizing antibody responses and confer enhanced cross-protection.

The prevalence of porcine reproductive and respiratory syndrome virus 1 (PRRSV1) isolates has continued to increase in Chinese swine herds in recent years. However, no effective control strategy is available for PRRSV1 infection in China. In this study, we generated the first infectious cDNA clone (rHLJB1) of a Chinese PRRSV1 isolate and subsequently used it as a backbone to construct an ORF2-6 chimeric virus (ORF2-6-CON). This virus contained a synthesized consensus sequence of the PRRSV1 ORF2-6 gene encoding all the envelope proteins. The ORF2-6 consensus sequence shared > 90% nucleotide similarity with four representative strains (Amervac, BJEU06-1, HKEU16 and NMEU09-1) of PRRSV1 in China. ORF2-6-CON had replication efficacy similar to that of the backbone rHLJB1 virus in primary alveolar macrophages (PAMs) and exhibited cell tropism in Marc-145 cells. Piglet inoculation and challenge studies indicated that ORF2-6-CON is not pathogenic to piglets and can induce enhanced cross-protection against a heterologous SD1291 isolate. Notably, ORF2-6-CON inoculation induced higher levels of heterologous neutralizing antibodies (nAbs) against SD1291 than rHLJB1 inoculation, which was concurrent with a higher percentage of T follicular helper (Tfh) cells in tracheobronchial lymph nodes (TBLNs), providing the first clue that porcine Tfh cells are correlated with heterologous PRRSV nAb responses. The number of SD1291-strain-specific IFNγ-secreting cells was similar in ORF2-6-CON-inoculated and rHLJB1-inoculated pigs. Overall, our findings support that the Marc-145-adapted ORF2-6-CON can trigger Tfh cell and heterologous nAb responses to confer improved cross-protection and may serve as a candidate strain for the development of a cross-protective PRRSV1 vaccine.

Fc N-glycosylation of autoreactive Aß antibodies as a blood-based biomarker for Alzheimer's disease.

INTRODUCTION: Naturally occurring autoantibodies (nAbs) against the pathologic isoform of amyloid beta (Aß42 ) were found in body fluids and indicate a systemic B cell response that may prevent Alzheimer's disease (AD) onset. N-glycans attached to immunoglobulin G-Fab/Fc fragments are features that influence their mechanism of action. The aim was to study the role of N-glycans in nAbs-Aß42 . METHODS: nAbs-Aß42 were isolated from AD patients and age-/sex-matched controls (n = 40) and immunoglobulin preparations. Glycosylated/deglycosylated nAbs-Aß42 were analyzed for their effect on Aß42 's aggregation, toxicity, and phagocytosis. Glycan structure was analyzed using matrix assisted laser desorption ionization time of flight mass spectrometry. RESULTS: Deglycosylation of nAbs-Aß42 had a major impact on Aß42 's aggregation/toxicity/phagocytosis. The glycan structure showed considerable differences between AD and controls. We were able to predict disease status with a sensitivity/specificity of 95% (confidence interval [CI]: 76.4-99.7%)/100% (CI: 83.9-100%). DISCUSSION: N-glycosylation has been identified as a critical attribute maintaining the beneficial effects of autoreactive Aß antibodies. These data have consequences for the development of monocloncal Aß antibodies and may open new avenues for diagnostics.

Variants of SARS-CoV-2: Influences on the Vaccines' Effectiveness and Possible Strategies to Overcome Their Consequences.

The immune response elicited by the current COVID-19 vaccinations declines with time, especially among the immunocompromised population. Furthermore, the emergence of novel SARS-CoV-2 variants, particularly the Omicron variant, has raised serious concerns about the efficacy of currently available vaccines in protecting the most vulnerable people. Several studies have reported that vaccinated people get breakthrough infections amid COVID-19 cases. So far, five variants of concern (VOCs) have been reported, resulting in successive waves of infection. These variants have shown a variable amount of resistance towards the neutralising antibodies (nAbs) elicited either through natural infection or the vaccination. The spike (S) protein, membrane (M) protein, and envelope (E) protein on the viral surface envelope and the N-nucleocapsid protein in the core of the ribonucleoprotein are the major structural vaccine target proteins against COVID-19. Among these targets, S Protein has been extensively exploited to generate effective vaccines against COVID-19. Hence, amid the emergence of novel variants of SARS-CoV-2, we have discussed their impact on currently available vaccines. We have also discussed the potential roles of S Protein in the development of novel vaccination approaches to contain the negative consequences of the variants' emergence and acquisition of mutations in the S Protein of SARS-CoV-2. Moreover, the implications of SARS-CoV-2's structural proteins were also discussed in terms of their variable potential to elicit an effective amount of immune response.

Mortality reduction in pediatric patients with severe fatal human adenoviral pneumonia treated with high titer neutralizing antibodies (NAbs) plasma: a retrospective cohort study.

BACKGROUND: Severe fatal human adenoviral (HAdV) pneumonia is associated with significant mortality and no effective drug is available for clinical therapy. We evaluated the association and safety of high titer neutralizing antibodies (NAbs) plasma in pediatric patients with severe fatal HAdV pneumonia. METHODS: A retrospective cohort study was performed between January 2016 to June 2021 in pediatric intensive care unit. Pediatric patients with severe fatal HAdV pneumonia were included and divided into plasma group (conventional treatment plus high titer NAbs plasma treatment) and control group (conventional treatment alone). The primary outcome was mortality in hospital. Secondary outcomes were the duration of fever after adenovirus genotype determined, duration of invasive mechanical ventilation, length of hospital stay. T-test, Mann-Whitney U-test, chi-square test, univariable and multivariable logistic regression analysis, Kaplan-Meier method and log-rank test were adopted to compare differences between two groups. RESULTS: A total of 59 pediatric patients with severe fatal HAdV pneumonia were enrolled. They were divided into plasma group (n = 33) and control group (n = 26). The mortality in hospital was 28.8% (17/ 59). Significantly fewer patients progressed to death in plasma group than control group (18.2% vs 42.3%, p = 0.042). Sequential organ failure assessment (SOFA) score, oxygen index (OI) and high titer NAbs plasma treatment were included in multivariable logistic regression analysis for mortality risk factors. Consequentially, SOFA score (Hazard Ratio [HR] 7.686, 95% Confidence Interval [CI] 1.735-34.054, p = 0.007) and without high titer NAbs plasma treatment (HR 4.298, 95%CI 1.030-17.934, p = 0.045) were significantly associated with mortality. In addition, high titer NAbs plasma treatment were associated with faster temperature recovering in survivors (p = 0.031). No serious adverse effects occurred. CONCLUSIONS: Administration of high titer NAbs plasma were associated with a lower hazard for mortality in pediatric patients with severe fatal HAdV pneumonia. For survivors, high titer NAbs plasma treatment shorten the duration of fever.

Alpha-Synuclein Autoimmune Decline in Prodromal Multiple System Atrophy and Parkinson's Disease.

Multiple-system trophy (MSA) and Parkinson's Disease (PD) are both progressive, neurodegenerative diseases characterized by neuropathological deposition of aggregated alpha-synuclein (αSyn). The causes behind this aggregation are still unknown. We have reported aberrancies in MSA and PD patients in naturally occurring autoantibodies (nAbs) against αSyn (anti-αSyn-nAbs), which are important partakers in anti-aggregatory processes, immune-mediated clearance, and anti-inflammatory functions. To elaborate further on the timeline of autoimmune aberrancies towards αSyn, we investigated here the Immunoglobulin (Ig) affinity profile and subclass composition (IgG-total, IgG1-4 and IgM) of anti-αSyn-nAbs in serum samples from prodromal (p) phases of MSA and PD. Using an electrochemiluminescence competition immunoassay, we confirmed that the repertoire of high-affinity anti-αSyn-nAbs is significantly reduced in pMSA and pPD. Further, we demonstrated that pPD had increased anti-αSyn IgG-total levels compared to pMSA and controls, concordant with increased anti-αSyn IgG1 levels in pPD. Anti-αSyn IgG2 and IgG4 levels were reduced in pMSA and pPD compared with controls, whereas anti-αSyn IgG3 levels were reduced in pMSA compared to pPD and controls. The results indicate that the impaired reactivity towards αSyn occurs prior to disease onset. The apparent lack of high-affinity anti-αSyn nAbs may result in reduced clearance of αSyn, leading to aggregation of the protein. Thus, this study provides novel insights into possible causes behind the pathogenesis in synucleinopathies such as MSA and PD.

HCV Glycoprotein Structure and Implications for B-Cell Vaccine Development.

Despite the approval of highly efficient direct-acting antivirals in the last decade Hepatitis C virus (HCV) remains a global health burden and the development of a vaccine would constitute an important step towards the control of HCV. The high genetic variability of the viral glycoproteins E1 and E2, which carry the main neutralizing determinants, together with their intrinsic structural flexibility, the high level of glycosylation that shields conserved neutralization epitopes and immune evasion using decoy epitopes renders the design of an efficient vaccine challenging. Recent structural and functional analyses have highlighted the role of the CD81 receptor binding site on E2, which overlaps with those neutralization epitopes within E2 that have been structurally characterized to date. This CD81 binding site consists of three distinct segments including "epitope I", "epitope II" and the "CD81 binding loop". In this review we summarize the structural features of the HCV glycoproteins that have been derived from X-ray structures of neutralizing and non-neutralizing antibody fragments complexed with either recombinant E2 or epitope-derived linear peptides. We focus on the current understanding how neutralizing antibodies interact with their cognate antigen, the structural features of the respective neutralization epitopes targeted by nAbs and discuss the implications for informed vaccine design.

IgG Fc N-glycosylation: Alterations in neurologic diseases and potential therapeutic target?

Immunoglobulin G (IgG) is the most abundant antibody subclass of the human circulatory system and has important functions in the adaptive immune response. On the one hand, recognition and neutralization of antigens is mediated by the fab fragment, and on the other hand, processes such as phagocytosis, complement activation and inflammatory reactions are triggered by the Fc fragment. Here, the composition of conserved N-glycans attached to asparagine 297 of the IgG CH2 domain is a major critical factor that particularly modulates the effector functions of IgG. Additional attachments of fucoses, galactoses, N-acetylglucosamines, and sialic acids have been identified as factors that influence the affinity to a wide range of complement proteins and receptors and, thus, secondarily induce the secretion of pro- and anti-inflammatory cytokines. Consequently, alterations in the IgG Fc N-glycosylation pattern can provoke disruptions in the immunological state and are accompanied by various diseases, although the involvement of changed IgG glycosylation in disease outbreaks remains unknown. In addition to many autoimmune diseases, which have already been extensively reviewed, there are a number of further disorders related to altered IgG glycosylation patterns. In the present review, we focus on neurologic diseases, as in the last few years, an increasing number of studies have been published in this field. Due to the absence of reliable early biomarkers as well as therapeutic options in many cases, such analyses are of great interest and reveal possible future approaches.

CoronaVac-vaccinated kidney transplant recipients with hybrid immunity have strong neutralizing responses against Omicron and Mu variants of SARS-CoV-2.

Neutralizing antibody (nAb) responses against SARS-CoV-2 variants after inactivated virus vaccine (CoronaVac) in kidney transplant recipients (KTRs) with or without SARS-CoV-2 infection history remains unclear. We aimed to evaluate the neutralizing antibody responses against emerging SARS-CoV-2 variants after two doses of CoronaVac in these patients. 22.2% of participants had hybrid immunity. Anti-spike IgG antibodies were evidenced in 44% of the patients. nAbs against B.1.111, Mu, and Omicron were detected in 28.5%, 17.9%, and 21.4% of naïve KTRs, respectively. Furthermore, nearly 100% of KTRs with hybrid immunity had nAbs against the variants evaluated. Thus, a significant proportion of infection-naïve KTRs had no detectable nAb titers against Mu and Omicron variants after two doses of the CoronaVac vaccine. However, the nAb titers were significantly higher in patients with hybrid immunity, and it was no association between the immunosuppressive regimen and the seropositivity rate of anti-SARS-CoV-2 neutralizing antibodies. Therefore, hybrid KTRs are protected against COVID-19 by emerging variants able to escape from vaccine-elicited nAbs such as Mu and Omicron.

Novel Competitive ELISA Utilizing Trimeric Spike Protein of SARS-CoV-2, Could Identify More Than RBD-RBM Specific Neutralizing Antibodies in Hybrid Sera.

Since the initiation of the COVID-19 pandemic, there has been a need for the development of diagnostic methods to determine the factors implicated in mounting an immune response against the virus. The most promising indicator has been suggested to be neutralizing antibodies (nAbs), which mainly block the interaction between the Spike protein (S) of SARS-CoV-2 and the host entry receptor ACE2. In this study, we aimed to develop and optimize conditions of a competitive ELISA to measure serum neutralizing titer, using a recombinant trimeric Spike protein modified to have six additional proline residues (S(6P)-HexaPro) and h-ACE2. The results of our surrogate Virus Neutralizing Assay (sVNA) were compared against the commercial sVNT (cPass, Nanjing GenScript Biotech Co., Nanjing City, China), using serially diluted sera from vaccinees, and a high correlation of ID50-90 titer values was observed between the two assays. Interestingly, when we tested and compared the neutralizing activity of sera from eleven fully vaccinated individuals who subsequently contracted COVID-19 (hybrid sera), we recorded a moderate correlation between the two assays, while higher sera neutralizing titers were measured with sVNA. Our data indicated that the sVNA, as a more biologically relevant model assay that paired the trimeric S(6P) with ACE2, instead of the isolated RBD-ACE2 pairing cPass test, could identify nAbs other than the RBD-RBM specific ones.

Predictive markers of safety and immunogenicity of adjuvanted vaccines.

Vaccination represents one of the greatest public health triumphs; in part due to the effect of adjuvants that have been included in vaccine preparations to boost the immune responses through different mechanisms. Although a variety of novel adjuvants have been under development, only a limited number have been approved by regulatory authorities for human vaccines. This report reflects the conclusions of a group of scientists from academia, regulatory agencies and industry who attended a conference on the current state of the art in the adjuvant field. Held at the U.S. Pharmacopeial Convention (USP) in Rockville, Maryland, USA, from 18 to 19 April 2013 and organized by the International Association for Biologicals (IABS), the conference focused particularly on the future development of effective adjuvants and adjuvanted vaccines and on overcoming major hurdles, such as safety and immunogenicity assessment, as well as regulatory scrutiny. More information on the conference output can be found on the IABS website, http://www.iabs.org/.

An Antibody-Dependent Cellular Cytotoxicity Assay for Detecting Ocrelizumab Neutralizing Antibody.

Ocrelizumab (OCREVUS®) is a humanized anti-CD20 monoclonal antibody approved for the treatment of adult patients with relapsing multiple sclerosis (RMS) and primary progressive multiple sclerosis (PPMS). Here, we discuss the strategic and technical considerations needed to develop a robust antibody-dependent cellular cytotoxicity (ADCC)-based neutralizing antibody (NAb) assay to detect anti-ocrelizumab NAb in patients enrolled in the ocrelizumab registered clinical trials. The NAb detection assay consisted of a two-tier assay that included a screening assay and a confirmation assay. In the screening assay, patient samples were analyzed in the presence of ocrelizumab. Samples that tested positive in the screening assay were subsequently analyzed in the confirmatory assay where another anti-CD20 mAb, obinutuzumab, was replaced by ocrelizumab, to verify NAb specificity. Both assays utilized MEC-2 cells, a chronic B cell leukemia cell line, pre-labeled with calcein AM as the target cells, and natural killer (NK) cells engineered to stably express Fc gamma receptor IIIa_ F158 as effector cells. Both cell lines were prepared to be thaw-and-use cells. The NAb assay measures fluorescence from the calcein AM released into the assay media upon the lysis of target cells by ADCC in the presence of ocrelizumab or obinutuzumab. Our validated NAb assay showed a relative sensitivity of 743 ng/mL and can detect 1500 ng/mL of a surrogate positive control antibody in the presence of 1500 ng/mL ocrelizumab. This ADCC assay is the first reported NAb assay that directly measures target cell lysis by using thaw-and-use target and effector cells simultaneously.

Duck hepatitis A virus type 1 transmission by exosomes establishes a productive infection in vivo and in vitro.

Duck hepatitis A virus type 1 (DHAV-1) infection causes an acute and highly fatal disease in young ducklings. Exosomes are nano-sized small extracellular vesicles secreted by various cells, which participate in intercellular communication and play a key role in the physiological and pathological processes. However, the role of exosomes in DHAV-1 transmission remains unknown. In this study, through RT-PCR, WB analysis and TEM observation, the complete DHAV-1 genomic RNA, partial viral proteins, and virions were respectively identified in the exosomes derived from DHAV-1-infected duck embryo fibroblasts (DEFs). The productive DHAV-1 infection was transmitted by exosomes in DEFs, duck embryos, and ducklings, and high titers of neutralizing antibodies completely blocked DHAV-1 infection but did not significantly neutralize exosome-mediated DHAV-1 infection. To the best of our knowledge, this is the first report that exosome-mediated DHAV-1 infection was resistant to antibody neutralization in vivo and in vitro, which might be an immune evasion mechanism of DHAV-1.

Longitudinal proteomic investigation of COVID-19 vaccination.

Although the development of COVID-19 vaccines has been a remarkable success, the heterogeneous individual antibody generation and decline over time are unknown and still hard to predict. In this study, blood samples were collected from 163 participants who next received two doses of an inactivated COVID-19 vaccine (CoronaVac®) at a 28-day interval. Using TMT-based proteomics, we identified 1,715 serum and 7,342 peripheral blood mononuclear cells (PBMCs) proteins. We proposed two sets of potential biomarkers (seven from serum, five from PBMCs) at baseline using machine learning, and predicted the individual seropositivity 57 days after vaccination (AUC = 0.87). Based on the four PBMC's potential biomarkers, we predicted the antibody persistence until 180 days after vaccination (AUC = 0.79). Our data highlighted characteristic hematological host responses, including altered lymphocyte migration regulation, neutrophil degranulation, and humoral immune response. This study proposed potential blood-derived protein biomarkers before vaccination for predicting heterogeneous antibody generation and decline after COVID-19 vaccination, shedding light on immunization mechanisms and individual booster shot planning.

Neonatal SHIV infection in rhesus macaques elicited heterologous HIV-1-neutralizing antibodies.

Infants and children infected with human immunodeficiency virus (HIV)-1 have been shown to develop neutralizing antibodies (nAbs) against heterologous HIV-1 strains, characteristic of broadly nAbs (bnAbs). Thus, having a neonatal model for the induction of heterologous HIV-1 nAbs may provide insights into the mechanisms of neonatal bnAb development. Here, we describe a neonatal model for heterologous HIV-1 nAb induction in pathogenic simian-HIV (SHIV)-infected rhesus macaques (RMs). Viral envelope (env) evolution showed mutations at multiple sites, including nAb epitopes. All 13 RMs generated plasma autologous HIV-1 nAbs. However, 8/13 (62%) RMs generated heterologous HIV-1 nAbs with increasing potency over time, albeit with limited breadth, and mapped to multiple nAb epitopes, suggestive of a polyclonal response. Moreover, plasma heterologous HIV-1 nAb development was associated with antigen-specific, lymph-node-derived germinal center activity. We define a neonatal model for heterologous HIV-1 nAb induction that may inform future pediatric HIV-1 vaccines for bnAb induction in infants and children.

Development of receptor binding domain-based double-antigen sandwich lateral flow immunoassay for the detection and evaluation of SARS-CoV-2 neutralizing antibody in clinical sera samples compared with the conventional virus neutralization test.

Vaccination is an effective strategy to fight COVID-19. However, the effectiveness of the vaccine varies among different populations in varying immune effects. Neutralizing antibody (NAb) level is an important indicator to evaluate the protective effect of immune response after vaccination. Lateral flow immunoassay (LFIA) is a rapid, safe and sensitivity detection method, which has great potential in the detection of SARS-CoV-2 NAb. In this study, a fluorescent beads-based lateral flow immunoassay (FBs-LFIA) and a latex beads-based LFIA (LBs-LFIA) using double antigen sandwich (DAS) strategy were established to detect NAbs in the serum of vaccinated people. The limit of detection (LoD) of the FBs-LFIA was 1.13 ng mL- 1 and the LBs-LFIA was 7.11 ng mL- 1. The two LFIAs were no cross-reactive with sera infected by other pathogenic bacteria. Furthermore, the two LFIAs showed a good performance in testing clinical samples. The sensitivity of FBs-LFIA and LBs-LFIA were 97.44% (95%CI: 93.15%-99.18%) and 98.29% (95%CI: 95.84%-99.37%), and the specificity were 98.28% (95%CI: 95.37%-99.45%) and 97.70% (95%CI: 94.82%-99.06%) compared with the conventional virus neutralization test (cVNT), respectively. Notably, the LBs-LFIA was also suitable for whole blood sample, requiring only 3 µL of whole blood, which provided the possibility to detect NAbs at home. To sum up, the two LFIAs based on double antigen sandwich established by us can rapidly, safely, sensitively and accurately detect SARS-CoV-2 NAb in human serum.

Plasma therapy: a passive resistance against the deadliest.

Convalescent plasma therapy provides a useful therapeutic tool to treat infectious diseases, especially where no specific therapeutic strategies have been identified. The ongoing pandemic puts back the spotlight on this age-old method as a viable treatment option. In this review, we discuss the usage of this therapy in different diseases including COVID-19, and the possible mechanisms of action. The current review also discusses the progress of therapeutic applications of blood-derivatives, from the simple transfer of immunized animal sera, to the more target-specific intravenous administration of human immunoglobulins from a pool of convalescent individuals, in both infectious and non-infectious diseases of various etiologies.

Plasma Metabolomic Alterations Induced by COVID-19 Vaccination Reveal Putative Biomarkers Reflecting the Immune Response.

Vaccination is currently the most effective strategy for the mitigation of the COVID-19 pandemic. mRNA vaccines trigger the immune system to produce neutralizing antibodies (NAbs) against SARS-CoV-2 spike proteins. However, the underlying molecular processes affecting immune response after vaccination remain poorly understood, while there is significant heterogeneity in the immune response among individuals. Metabolomics have often been used to provide a deeper understanding of immune cell responses, but in the context of COVID-19 vaccination such data are scarce. Mass spectrometry (LC-MS) and nuclear magnetic resonance (NMR)-based metabolomics were used to provide insights based on the baseline metabolic profile and metabolic alterations induced after mRNA vaccination in paired blood plasma samples collected and analysed before the first and second vaccination and at 3 months post first dose. Based on the level of NAbs just before the second dose, two groups, "low" and "high" responders, were defined. Distinct plasma metabolic profiles were observed in relation to the level of immune response, highlighting the role of amino acid metabolism and the lipid profile as predictive markers of response to vaccination. Furthermore, levels of plasma ceramides along with certain amino acids could emerge as predictive biomarkers of response and severity of inflammation.

Application and Validation of SARS-CoV-2 RBD Neutralizing ELISA Assay.

The establishment of an approach for detecting the anti-severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2)-receptor-binding domain (RBD) neutralizing antibodies (nAbs) by a safe, easy, and rapid technique without requiring the use of live viruses is essential for facing the coronavirus disease 2019 (COVID-19) pandemic. Depending on competitive enzyme-linked immunosorbent assay (ELISA) methodology, the current study assay was designed to simulate the virus-host interaction using purified SARS-COV-2-RBD from the spike protein and the host cell receptor human angiotensin-converting enzyme 2 protein. The performance of this in-house neutralizing ELISA assay was validated using freshly prepared standards with different known concentrations of the assay. In this regard, a cohort of 50 serum samples from convalescent COVID-19 individuals with different disease severity at different time points post-recovery and a cohort of 50 serum samples from healthy individuals were processed by the in-house developed assay for detecting SARS-CoV-2 nAbs, in comparison with a commercial total anti-SARS-CoV-2 IgG antibody assay as a gold standard. The assay obtained a sensitivity of 88% (95% CI: 75.69-95.47) and a specificity of 92% (95% CI: 80.77- 97.78%). A negative strong correlation was demonstrated in the standard curve between the optical density absorbance and log concentration of the nAbs with a statistical measure of r2 (coefficient of determination) = 0.9539. The SARS-COV-2-RBD neutralizing ELISA assay serves as a high throughput qualitative and quantitative tool that can be applied in most laboratory settings without special biosafety requirements to detect anti-RBD nAbs for seroprevalence, pre-clinical, and clinical evaluation of COVID-19 vaccines efficiency and the rapid selection of convalescent plasma donors for the treatment of COVID-19 patients.

Systemic and local immune responses to intraocular AAV vector administration in non-human primates.

Positive clinical outcomes in adeno-associated virus (AAV)-mediated retinal gene therapy have often been attributed to the low immunogenicity of AAVs and immune privilege of the eye. However, several recent studies have shown potential for inflammatory responses. The current understanding of the factors contributing to inflammation, such as the pre-existence of serum antibodies against AAVs and their contribution to increases in antibody levels post-injection, is incomplete. The parameters that regulate the generation of new antibodies in response to the AAV capsid or transgene after intraocular injections are also insufficiently described. This study is a retrospective analysis of the pre-existing serum antibodies in correlation with changes in antibody levels after intraocular injections of AAV in non-human primates (NHPs) of the species Macaca fascicularis. In NHP serums, we analyzed the binding antibody (BAB) levels and a subset of these called neutralizing antibodies (NABs) that impede AAV transduction. We observed significantly higher pre-existing serum BABs against AAV8 compared with other serotypes and a dose-dependent increase in BABs and NABs in the serums collected post-injection, irrespective of the serotype or the mode of injection. Lastly, we were able to demonstrate a correlation between the serum BAB levels with clinical grading of inflammation and levels of transgene expression.