Protocol for structural modeling of antibody to human leukocyte antigen interaction using discovery and targeted cross-linking mass spectrometry.

Cross-linking mass spectrometry (XL-MS) provides low-resolution structural information to model protein structures. Here, we present a protocol to identify cross-links of purified antibody binding to purified human leukocyte antigen (HLA). We describe steps for using a discovery-based XL-MS approach followed by a targeted XL-MS approach. We then detail procedures for using the identified cross-links with other structural data for molecular docking of the antibody to HLA. This protocol has applications for modeling the interacting structure of purified antibody to antigen. For complete details on the use and execution of this protocol, please refer to Ser et al.1.

Relative deficiency in interferon-γ-secreting CD4+ T cells is strongly associated with poorer COVID-19 vaccination responses in older adults.

Although the two-dose mRNA vaccination regime provides protection against SARS-CoV-2, older adults have been shown to exhibit poorer vaccination responses. In addition, the role of vaccine-induced T-cell responses is not well characterised. We aim to assess the impact of age on immune responses after two doses of the BNT162b2 mRNA vaccine, focussing on antigen-specific T-cells. A prospective 3-month study was conducted on 15 young (median age 31 years, interquartile range (IQR) 25-35 years) and 14 older adults (median age 72 years, IQR 70-73 years). We assessed functional, neutralising antibody responses against SARS-CoV-2 variants using ACE-2 inhibition assays, and changes in B and T-cell subsets by high-dimensional flow cytometry. Antigen-specific T-cell responses were also quantified by intracellular cytokine staining and flow cytometry. Older adults had attenuated T-helper (Th) response to vaccination, which was associated with weaker antibody responses and decreased SARS-CoV-2 neutralisation. Antigen-specific interferon-γ (IFNγ)-secreting CD4+ T-cells to wild-type and Omicron antigens increased in young adults, which was strongly positively correlated with their neutralising antibody responses. Conversely, this relationship was negative in older adults. Hence, older adults' relative IFNγ-secreting CD4+ T cell deficiency might explain their poorer COVID-19 vaccination responses. Further exploration into the aetiology is needed and would be integral in developing novel vaccination strategies and improving infection outcomes in older adults.

Durable cross-protective neutralizing antibody responses elicited by lipid nanoparticle-formulated SARS-CoV-2 mRNA vaccines.

The advent of SARS-CoV-2 variants with defined mutations that augment pathogenicity and/or increase immune evasiveness continues to stimulate global efforts to improve vaccine formulation and efficacy. The extraordinary advantages of lipid nanoparticles (LNPs), including versatile design, scalability, and reproducibility, make them ideal candidates for developing next-generation mRNA vaccines against circulating SARS-CoV-2 variants. Here, we assess the efficacy of LNP-encapsulated mRNA booster vaccines encoding the spike protein of SARS-CoV-2 for variants of concern (Delta, Omicron) and using a predecessor (YN2016C isolated from bats) strain spike protein to elicit durable cross-protective neutralizing antibody responses. The mRNA-LNP vaccines have desirable physicochemical characteristics, such as small size (~78 nm), low polydispersity index (<0.13), and high encapsulation efficiency (>90%). We employ in vivo bioluminescence imaging to illustrate the capacity of our LNPs to induce robust mRNA expression in secondary lymphoid organs. In a BALB/c mouse model, a three-dose subcutaneous immunization of mRNA-LNPs vaccines achieved remarkably high levels of cross-neutralization against the Omicron B1.1.529 and BA.2 variants for extended periods of time (28 weeks) with good safety profiles for all constructs when used in a booster regime, including the YN2016C bat virus sequences. These findings have important implications for the design of mRNA-LNP vaccines that aim to trigger durable cross-protective immunity against the current and newly emerging variants.

Type I Interferon Autoantibodies Correlate With Cellular Immune Alterations in Severe COVID-19.

BACKGROUND: Infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can lead to severe disease with increased morbidity and mortality among certain risk groups. The presence of autoantibodies against type I interferons (aIFN-Abs) is one mechanism that contributes to severe coronavirus disease 2019 (COVID-19). METHODS: This study aimed to investigate the presence of aIFN-Abs in relation to the soluble proteome, circulating immune cell numbers, and cellular phenotypes, as well as development of adaptive immunity. RESULTS: aIFN-Abs were more prevalent in critical compared to severe COVID-19 but largely absent in the other viral and bacterial infections studied here. The antibody and T-cell response to SARS-CoV-2 remained largely unaffected by the presence aIFN-Abs. Similarly, the inflammatory response in COVID-19 was comparable in individuals with and without aIFN-Abs. Instead, presence of aIFN-Abs had an impact on cellular immune system composition and skewing of cellular immune pathways. CONCLUSIONS: Our data suggest that aIFN-Abs do not significantly influence development of adaptive immunity but covary with alterations in immune cell numbers.

Employment of a high throughput functional assay to define the critical factors that influence vaccine induced cross-variant neutralizing antibodies for SARS-CoV-2.

The scale and duration of neutralizing antibody responses targeting SARS-CoV-2 viral variants represents a critically important serological parameter that predicts protective immunity for COVID-19. In this study, we describe the development and employment of a new functional assay that measures neutralizing antibodies for SARS-CoV-2 and present longitudinal data illustrating the impact of age, sex and comorbidities on the kinetics and strength of vaccine-induced antibody responses for key variants in an Asian volunteer cohort. We also present an accurate quantitation of serological responses for SARS-CoV-2 that exploits a unique set of in-house, recombinant human monoclonal antibodies targeting the viral Spike and nucleocapsid proteins and demonstrate a reduction in neutralizing antibody titres across all groups 6 months post-vaccination. We also observe a marked reduction in the serological binding activity and neutralizing responses targeting recently newly emerged Omicron variants including XBB 1.5 and highlight a significant increase in cross-protective neutralizing antibody responses following a third dose (boost) of vaccine. These data illustrate how key virological factors such as immune escape mutations combined with host demographic factors such as age and sex of the vaccinated individual influence the strength and duration of cross-protective serological immunity for COVID-19.

Hybrid structural modeling of alloantibody binding to human leukocyte antigen with rapid and reproducible cross-linking mass spectrometry.

Alloantibody recognition of donor human leukocyte antigen (HLA) is associated with poor clinical transplantation outcomes. However, the molecular and structural basis for the alloantibody-HLA interaction is not well understood. Here, we used a hybrid structural modeling approach on a previously studied alloantibody-HLA interacting pair with inputs from ab initio, in silico, and in vitro data. Highly reproducible cross-linking mass spectrometry data were obtained with both discovery- and targeted mass spectrometry-based approaches approaches. The cross-link information was then used together with predicted antibody Fv structure, predicted antibody paratope, and in silico-predicted interacting surface to model the antibody-HLA interaction. This hybrid structural modeling approach closely recapitulates the key interacting residues from a previously solved crystal structure of an alloantibody-HLA-A∗11:01 pair. These results suggest that a predictive-based hybrid structural modeling approach supplemented with cross-linking mass spectrometry data can provide functionally relevant structural models to understand the structural basis of antibody-HLA mismatch in transplantation.

An anti-LpqH human monoclonal antibody from an asymptomatic individual mediates protection against Mycobacterium tuberculosis.

Tuberculosis (TB) is an airborne disease caused by Mycobacterium tuberculosis (Mtb). Whilst a functional role for humoral immunity in Mtb protection remains poorly defined, previous studies have suggested that antibodies can contribute towards host defense. Thus, identifying the critical components in the antibody repertoires from immune, chronically exposed, healthy individuals represents an approach for identifying new determinants for natural protection. In this study, we performed a thorough analysis of the IgG/IgA memory B cell repertoire from occupationally exposed, immune volunteers. We detail the identification and selection of a human monoclonal antibody that exhibits protective activity in vivo and show that it targets a virulence factor LpqH. Intriguingly, protection in both human ex vivo and murine challenge experiments was isotype dependent, with most robust protection being mediated via IgG2 and IgA. These data have important implications for our understanding of natural mucosal immunity for Mtb and highlight a new target for future vaccine development.

Atypical B cells and impaired SARS-CoV-2 neutralization following heterologous vaccination in the elderly.

Suboptimal responses to a primary vaccination course have been reported in the elderly, but there is little information regarding the impact of age on responses to booster third doses. Here, we show that individuals 70 years or older (median age 73, range 70-75) who received a primary two-dose schedule with AZD1222 and booster third dose with mRNA vaccine achieve significantly lower neutralizing antibody responses against SARS-CoV-2 spike pseudotyped virus compared with those younger than 70 (median age 66, range 54-69) at 1 month post booster. Impaired neutralization potency and breadth post third dose in the elderly is associated with circulating "atypical" spike-specific B cells expressing CD11c and FCRL5. However, when considering individuals who received three doses of mRNA vaccine, we did not observe differences in neutralization or enrichment in atypical B cells. This work highlights the finding that AdV and mRNA COVID-19 vaccine formats differentially instruct the memory B cell response.

CD28-CAR-T cell activation through FYN kinase signaling rather than LCK enhances therapeutic performance.

Signal transduction induced by chimeric antigen receptors (CARs) is generally believed to rely on the activity of the SRC family kinase (SFK) LCK, as is the case with T cell receptor (TCR) signaling. Here, we show that CAR signaling occurs in the absence of LCK. This LCK-independent signaling requires the related SFK FYN and a CD28 intracellular domain within the CAR. LCK-deficient CAR-T cells are strongly signaled through CAR and have better in vivo efficacy with reduced exhaustion phenotype and enhanced induction of memory and proliferation. These distinctions can be attributed to the fact that FYN signaling tends to promote proliferation and survival, whereas LCK signaling promotes strong signaling that tends to lead to exhaustion. This non-canonical signaling of CAR-T cells provides insight into the initiation of both TCR and CAR signaling and has important clinical implications for improvement of CAR function.

Efficient recall of SARS-CoV-2 variant-reactive B cells and T responses in the elderly upon heterologous mRNA vaccines as boosters.

Waning antibody levels against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the emergence of variants of concern highlight the need for booster vaccinations. This is particularly important for the elderly population, who are at a higher risk of developing severe coronavirus disease 2019 (COVID-19) disease. While studies have shown increased antibody responses following booster vaccination, understanding the changes in T and B cell compartments induced by a third vaccine dose remains limited. We analyzed the humoral and cellular responses in subjects who received either a homologous messenger RNA(mRNA) booster vaccine (BNT162b2 + BNT162b2 + BNT162b2; ''BBB") or a heterologous mRNA booster vaccine (BNT162b2 + BNT162b2 + mRNA-1273; ''BBM") at Day 0 (prebooster), Day 7, and Day 28 (postbooster). Compared with BBB, elderly individuals (≥60 years old) who received the BBM vaccination regimen display higher levels of neutralizing antibodies against the Wuhan and Delta strains along with a higher boost in immunoglobulin G memory B cells, particularly against the Omicron variant. Circulating T helper type 1(Th1), Th2, Th17, and T follicular helper responses were also increased in elderly individuals given the BBM regimen. While mRNA vaccines increase antibody, T cell, and B cell responses against SARS-CoV-2 1 month after receiving the third dose booster, the efficacy of the booster vaccine strategies may vary depending on age group and regimen combination.

SARS-CoV-2 Antibody Effectiveness Is Influenced by Non-Epitope Mutation/Binding-Induced Denaturation of the Epitope 3D Architecture.

The public health threat from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to intensify with emerging variants of concern (VOC) aiming to render COVID-19 vaccines/infection-induced antibodies redundant. The SARS-CoV-2 spike protein is responsible for receptor binding and infection of host cells making it a legitimate antibody target. Antibodies mostly target epitopes in the receptor binding domain (RBD). Mutations occurring within epitopes influence antibody specificity and function by altering their 3D architecture. However, the mechanisms by which non-epitope mutations in the RBD influence antibody specificity and function remain a mystery. We used Protein Data Bank (PDB) deposited 3D structures for the original, Beta, Delta, BA.1, and BA.2 RBD proteins in complex with either neutralizing antibodies or Angiotensin-Converting Enzyme 2 (ACE2) to elucidate the structural and mechanistic basis for neutralizing antibody evasion driven by non-epitope amino acid substitutions in the RBD. Since the mechanism behind the extensively reported functional discrepancies between the same antibody when used individually and when used in an antibody cocktail is lacking, we explored the structural basis for this inconsistency. Finally, since SARS-CoV-2 antibodies are viral mutagens, we deciphered determinants for antibody-pressured amino acid substitutions. On the one hand, we show that non-epitope mutations in the RBD domain of SARS-CoV-2 VOC influence the formation of hydrogen bonds in the paratope-epitope interface by repositioning RBD amino-acid sidechains (AASCs). This increases the distance between complementary donor/acceptor atoms on paratope and epitope AASCs leading to weaker or the complete prevention of the formation of hydrogen bonds in the paratope-epitope interface. On the other hand, we show that SARS-CoV-2 VOC employ the same strategy to simultaneously search for complementary donor/acceptor atoms on ACE2 AASCs to form new interactions, potentially favoring increased viral transmission. Additionally, we illustrate that converting the spike protein to an RBD, a deletion mutation, also repositions epitope AASCs and that AASC interactions in the paratope-epitope interface vary when an antibody is used individually versus when utilized as a cocktail with other antibodies. Finally, we show that the process of substituting immunogenic RBD amino acids begins with the repositioning of their AASCs induced by immune/antibody pressure. We show that donor/acceptor atoms from any amino acid can determine cross-reactivity instead, provided they possess and present spatially pairing donor/acceptor atoms. By studying structural alignments for PDB deposited antibody-RBD 3D structures and relating them to published binding and neutralization profiles of the same antibodies, we demonstrate that minor structural alterations such as epitope AASC repositioning have a major impact on antibody effectiveness and, hence, should receive adequate attention given that protein structure dictates protein function.

Defining the specificity and function of a human neutralizing antibody for Hepatitis B virus.

Hepatitis B Virus (HBV) is a hepadnavirus that is the principal pathogen underlying viral liver disease in human populations. In this study, we describe the isolation and characterization of a fully human monoclonal antibody for HBV. This HuMab was isolated by a combinatorial screen of the memory B-cell repertoire from an acute/recovered HBV-infected patient. Lead candidate selection was based upon strong binding and neutralizing activity for live HBV. We provide a detailed biochemical/biophysical, and subclass characterization of its specificity and affinity against all of the principal HBV genotypes combined with a functional analysis of its in vitro activity. We also demonstrate its potential as a prophylaxis/therapy in vivo using human liver chimeric mouse models for HBV infection. These data have important implications for our understanding of natural human immunity to HBV and suggest that this potentially represents a new antibody-based anti-viral candidate for prophylaxis and/or therapy for HBV infection.

Immune and pathophysiologic profiling of antenatal coronavirus disease 2019 in the GIFT cohort: A Singaporean case-control study.

COVID-19 can be severe in pregnant women, and have adverse consequences for the subsequent infant. We profiled the post-infectious immune responses in maternal and child blood as well as breast milk in terms of antibody and cytokine expression and performed histopathological studies on placentae obtained from mothers convalescent from antenatal COVID-19. Seventeen mother-child dyads (8 cases of antenatal COVID-19 and 9 healthy unrelated controls; 34 individuals in total) were recruited to the Gestational Immunity For Transfer (GIFT) study. Maternal and infant blood, and breast milk samples were collected over the first year of life. All samples were analyzed for IgG and IgA against whole SARS-CoV-2 spike protein, the spike receptor-binding domain (RBD), and previously reported immunodominant epitopes, as well as cytokine levels. The placentae were examined microscopically. The study is registered at clinicaltrials.gov under the identifier NCT04802278. We found high levels of virus-specific IgG in convalescent mothers and similarly elevated titers in newborn children. Thus, antenatal SARS-CoV-2 infection led to high plasma titers of virus-specific antibodies in infants postnatally. However, this waned within 3-6 months of life. Virus neutralization by plasma was not uniformly achieved, and the presence of antibodies targeting known immunodominant epitopes did not assure neutralization. Virus-specific IgA levels were variable among convalescent individuals' sera and breast milk. Antibody transfer ratios and the decay of transplacentally transferred virus-specific antibodies in neonatal circulation resembled that for other pathogens. Convalescent mothers showed signs of chronic inflammation marked by persistently elevated IL17RA levels in their blood. Four placentae presented signs of acute inflammation, particularly in the subchorionic region, marked by neutrophil infiltration even though > 50 days had elapsed between virus clearance and delivery. Administration of a single dose of BNT162b2 mRNA vaccine to mothers convalescent from antenatal COVID-19 increased virus-specific IgG and IgA titers in breast milk, highlighting the importance of receiving the vaccine even after natural infection with the added benefit of enhanced passive immunity.

Lower vaccine-acquired immunity in the elderly population following two-dose BNT162b2 vaccination is alleviated by a third vaccine dose.

Understanding the impact of age on vaccinations is essential for the design and delivery of vaccines against SARS-CoV-2. Here, we present findings from a comprehensive analysis of multiple compartments of the memory immune response in 312 individuals vaccinated with the BNT162b2 SARS-CoV-2 mRNA vaccine. Two vaccine doses induce high antibody and T cell responses in most individuals. However, antibody recognition of the Spike protein of the Delta and Omicron variants is less efficient than that of the ancestral Wuhan strain. Age-stratified analyses identify a group of low antibody responders where individuals ≥60 years are overrepresented. Waning of the antibody and cellular responses is observed in 30% of the vaccinees after 6 months. However, age does not influence the waning of these responses. Taken together, while individuals ≥60 years old take longer to acquire vaccine-induced immunity, they develop more sustained acquired immunity at 6 months post-vaccination. A third dose strongly boosts the low antibody responses in the older individuals against the ancestral Wuhan strain, Delta and Omicron variants.

Association of systemic vitamin D on the course of dengue virus infection in adults: a single-centre dengue cohort study at a large institution in Singapore.

INTRODUCTION: Host immune responses may impact dengue severity in adults. Vitamin D has multiple immunomodulatory effects on innate and adaptive immunity. METHODS: We evaluated the association between systemic 25-hydroxyvitamin D [25-(OH) D] and dengue disease severity in adults. We measured plasma for total 25-(OH) D levels with an electrochemiluminescence immunoassay using stored samples from participants with laboratory confirmed dengue who were prospectively enrolled in 2012-2016 at our institution. RESULTS: 80 participants (median age 43 years) were enrolled. Six participants had severe dengue based on the World Health Organisation (WHO) 1997 criteria (i.e. dengue haemorrhagic fever/dengue shock syndrome) and another six had severe dengue based on the WHO 2009 criteria. Median 25-(OH) D at acute phase of dengue was 6.175 µg/L (interquartile range 3.82-8.21; range 3.00-15.29) in all participants. 25-(OH) D showed inverse linear trend with severe dengue manifestations based on the WHO 2009 criteria (aRR 0.72; 95% confidence interval 0.57-0.91; p < 0.01) after adjustment for age, gender and ethnicity. CONCLUSION: Limited studies have evaluated the role of systemic 25-(OH) D on dengue severity. Our study found low systemic 25-(OH) D was associated with increased dengue disease severity, particularly for severe bleeding that was not explained by thrombocytopenia. Further studies investigating the underlying immune mechanisms and effects on the vascular endothelium are needed.

Finger stick blood test to assess postvaccination SARS-CoV-2 neutralizing antibody response against variants.

There is clinical need for a quantifiable point-of-care (PoC) SARS-CoV-2 neutralizing antibody (nAb) test that is adaptable with the pandemic's changing landscape. Here, we present a rapid and semi-quantitative nAb test that uses finger stick or venous blood to assess the nAb response of vaccinated population against wild-type (WT), alpha, beta, gamma, and delta variant RBDs. It captures a clinically relevant range of nAb levels, and effectively differentiates prevaccination, post first dose, and post second dose vaccination samples within 10 min. The data observed against alpha, beta, gamma, and delta variants agrees with published results evaluated in established serology tests. Finally, our test revealed a substantial reduction in nAb level for beta, gamma, and delta variants between early BNT162b2 vaccination group (within 3 months) and later vaccination group (post 3 months). This test is highly suited for PoC settings and provides an insightful nAb response in a postvaccinated population.

Identification of B-Cell Epitopes for Eliciting Neutralizing Antibodies against the SARS-CoV-2 Spike Protein through Bioinformatics and Monoclonal Antibody Targeting.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global public health crisis. Effective COVID-19 vaccines developed by Pfizer-BioNTech, Moderna, and Astra Zeneca have made significant impacts in controlling the COVID-19 burden, especially in reducing the transmission of SARS-CoV-2 and hospitalization incidences. In view of the emergence of new SARS-CoV-2 variants, vaccines developed against the Wuhan strain were less effective against the variants. Neutralizing antibodies produced by B cells are a critical component of adaptive immunity, particularly in neutralizing viruses by blocking virus attachment and entry into cells. Therefore, the identification of protective linear B-cell epitopes can guide epitope-based peptide designs. This study reviews the identification of SARS-CoV-2 B-cell epitopes within the spike, membrane and nucleocapsid proteins that can be incorporated as potent B-cell epitopes into peptide vaccine constructs. The bioinformatic approach offers a new in silico strategy for the mapping and identification of potential B-cell epitopes and, upon in vivo validation, would be useful for the rapid development of effective multi-epitope-based vaccines. Potent B-cell epitopes were identified from the analysis of three-dimensional structures of monoclonal antibodies in a complex with SARS-CoV-2 from literature mining. This review provides significant insights into the elicitation of potential neutralizing antibodies by potent B-cell epitopes, which could advance the development of multi-epitope peptide vaccines against SARS-CoV-2.