Potent Neutralizing Research Articles

Combining computational modeling and experimental library screening to affinity-mature VEEV-neutralizing antibody F5.

Engineered monoclonal antibodies have proven to be highly effective therapeutics in recent viral outbreaks. However, despite technical advancements, an ability to rapidly adapt or increase antibody affinity and by extension, therapeutic efficacy, has yet to be fully realized. We endeavored to stand-up such a pipeline using molecular modeling combined with experimental library screening to increase the affinity of F5, a monoclonal antibody with potent neutralizing activity against Venezuelan Equine Encephalitis Virus (VEEV), to recombinant VEEV (IAB) E1E2 antigen. We modeled the F5/E1E2 binding interface and generated predictions for mutations to improve binding using a Rosetta-based approach and dTERMen, an informatics approach. The modeling was complicated by the fact that a high-resolution structure of F5 is not available and the H3 loop of F5 exceeds the length for which current modeling approaches can determine a unique structure. A subset of the predicted mutations from both methods were incorporated into a phage display library of scFvs. This library and a library generated by error-prone PCR were screened for binding affinity to the recombinant antigen. Results from the screens identified favorable mutations which were incorporated into 12 human-IgG1 variants. The best variant, containing eight mutations, improved KD from 0.63 nM (parental) to 0.01 nM. While this did not improve neutralization or therapeutic potency of F5 against IAB, it did increase cross-reactivity to other closely related VEEV epizootic and enzootic strains, demonstrating the potential of this method to rapidly adapt existing therapeutics to emerging viral strains.

Nanobody screening and machine learning guided identification of cross-variant anti-SARS-CoV-2 neutralizing heavy-chain only antibodies.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) continues to persist, demonstrating the risks posed by emerging infectious diseases to national security, public health, and the economy. Development of new vaccines and antibodies for emerging viral threats requires substantial resources and time, and traditional development platforms for vaccines and antibodies are often too slow to combat continuously evolving immunological escape variants, reducing their efficacy over time. Previously, we designed a next-generation synthetic humanized nanobody (Nb) phage display library and demonstrated that this library could be used to rapidly identify highly specific and potent neutralizing heavy chain-only antibodies (HCAbs) with prophylactic and therapeutic efficacy in vivo against the original SARS-CoV-2. In this study, we used a combination of high throughput screening and machine learning (ML) models to identify HCAbs with potent efficacy against SARS-CoV-2 viral variants of interest (VOIs) and concern (VOCs). To start, we screened our highly diverse Nb phage display library against several pre-Omicron VOI and VOC receptor binding domains (RBDs) to identify panels of cross-reactive HCAbs. Using HCAb affinity for SARS-CoV-2 VOI and VOCs (pre-Omicron variants) and model features from other published data, we were able to develop a ML model that successfully identified HCAbs with efficacy against Omicron variants, independent of our experimental biopanning workflow. This biopanning informed ML approach reduced the experimental screening burden by 78% to 90% for the Omicron BA.5 and Omicron BA.1 variants, respectively. The combined approach can be applied to other emerging viruses with pandemic potential to rapidly identify effective therapeutic antibodies against emerging variants.

Innovative production of highly potent equine neutralizing antibody against Hemiscorpius lepturus scorpion venom using recombinant mPLD1 protein.

Scorpion envenomation, especially from Hemiscorpius lepturus, poses a significant health risk, leading to considerable morbidity and mortality. The venom's major toxin, which includes phospholipase D (PLD), is responsible for various systemic complications. In prior studies, we identified a native phospholipase D (PLD) toxin as a key lethal factor in the venom of H. lepturus. A recombinant PLD that retained its toxicity was developed and designated as PLD1. Additionally, a non-toxic and devoid of lethal effects mutant form of the recombinant PLD1 protein, was produced and named as mPLD1. Building on this knowledge, we aimed to produce a novel antivenom using recombinant mPLD1-based immunogen and commercial antisera were included for comparison. Two horses were immunized separately with either recombinant or mutant PLD1, resulting in high titer antisera with no significant difference between the two immunogens. Purified F(ab')2 fragments derived from horse antisera demonstrated a markedly enhanced specificity in the detection of PLD1 and crude venom when compared to commercial alternatives. Furthermore, in vivo neutralization assays revealed that the antisera generated from mPLD1 protein was 89 and 36 times more potent than those of commercial ones. Horses produced highly neutralizing antibodies against PLD1 than the two local commercial antisera. These findings underscore the promise of the developed anti-mPLD1 as a highly effective therapeutic molecule for H. lepturus envenomation. Given that the production process for the recombinant immunogen is straightforward and utilizes cost-effective technologies, focusing on the manufacture of this highly efficient antisera could lead to significant advancements in horse antisera production platforms.

IgA class switching enhances neutralizing potency against SARS-CoV-2 by increased antibody hinge flexibility.

IgA antibodies are critical components of the mucosal immune barrier, providing essential first-line defense against viral infections. In this study, we investigated the impact of antibody class switching on neutralization efficacy by engineering recombinant antibodies of different isotypes (IgA1, IgG1) with identical variable regions from SARS-CoV-2 convalescent patients. A potent, broad-spectrum neutralizing monoclonal antibody CAV-C65 exhibited a ten-fold increase in neutralization potency upon switching from IgG1 to IgA1 monomer. Structural analysis revealed that this antibody binds to two adjacent receptor binding domains on the spike protein. Enhanced neutralization by IgA1 was attributed to the combined effects of increased affinity, unique hinge region properties, and potential cross-linking of viral particles. Inhaled CAV-C65 IgA1 demonstrated prophylactic efficacy against lethal SARS-CoV-2 infection in hACE2 mice. These findings highlight the pivotal role of IgA in antiviral immunity and inform the development of IgA-based therapeutics.

A type of cryptic epitope-binding antibody on SARS-CoV-2 RBD retains the neutralization against SARS-CoV-2 variants and sarbecoviruses.

The continuing emergence of SARS-CoV-2 variants has posed a great challenge to vaccination strategies. Therefore, the development of broad-spectrum protective antibodies and universal vaccines remains urgently needed. In this study, we isolated two broadly neutralizing mAbs, nCoV-R48 and nCoV-R70, from a vaccinated person. These two mAbs neutralize the SARS-CoV-2 prototype and variants, including Alpha, Beta, Gamma, Delta, BQ1.1, XBB, BF.7, and the recently emerged KP.2. Moreover, nCoV-R48 and nCoV-R70 also neutralize representative sarbecoviruses GD/1/2019 and SARS-CoV. We determined the epitopes of these two mAbs using X-ray crystallography and Cryo-EM. The structural analysis shows that nCoV-R48 and nCoV-R70 recognize the same cryptic epitope on the RBD. These antibodies belong to the RBD-8 class, which exhibits broad neutralizing potency by inducing S1 shedding. This cryptic epitope provides guidance for developing universal therapeutic antibodies against COVID-19.

Neutralizing antibody landscape of the non-polio Enteroviruses and future strategy.

The non-polio Enteroviruses (NPEVs), consist of enteroviruses, coxsackieviruses, echoviruses, and rhinoviruses, are causative agents for a wide variety of diseases, ranging from common cold to encephalitis and acute flaccid paralysis (AFP). In recent years, several NPEVs have become serious public health threats, include EV-A71, which has caused epidemics of hand-foot-and-mouth disease (HMFD) in Southeast Asia, and EV-D68, which caused outbreaks of severe respiratory disease in children worldwide. Infections with these viruses are associated with neurological diseases like aseptic meningitis and AFP. Currently, apart from inactivated EV-A71 vaccines that were developed in China, no effective measures are available to prevent or treat NPEV infections. Antibody-mediated immunity is crucial for preventing and limiting viral infections, and potent neutralizing antibodies could serve as potential therapeutic agents. In this review, we describe recent progress in the NPEVs neutralization antibodies, summarizing the characteristics, breadth, and potency against NPEVs, such as EV-A71, CVA16, EV-D68, and echovirus. We focus on not only through the study of viral epitopes but also through the understanding of virus-antibody interactions. Also, we decipher the role of antibodies in the attachment of the virus to receptors, internalization, and uncoating process, providing insight into virus neutralization mechanisms. Moreover, bi-specific antibodies or multivalent antibodies with better potency are also discussed. Therefore, an in-depth understanding of structures of enterovirus and mechanisms of antibody neutralization should be useful for future strategies in guiding the design of a rational antiviral agent against NPEVs infections.

Enhancing vaccine half-life as a novel strategy for improving immune response durability of subunit vaccines.

Vaccines are widely regarded as one of the most effective strategies for combating infectious diseases. However, significant challenges remain, such as insufficient antibody levels, limited protection against rapidly evolving variants, and poor immune durability, particularly in subunit vaccines, likely due to their short in vivo exposure. Recent advances in extending the half-life of protein therapeutics have shown promise in improving drug efficacy, yet whether increasing in vivo persistence can enhance the efficacy of subunit vaccines remains underexplored. In this study, we developed two trimeric SARS-CoV-2 subunit vaccines with distinct pharmacokinetic profiles to evaluate the impact of vaccine persistence on immune efficacy. A self-assembling trimeric subunit vaccine (RBD-HR/trimer) was designed, followed by an extended-persistence variant (RBD-sFc-HR/trimer) incorporating a soluble monomeric IgG1 fragment crystallizable. We demonstrated that RBD-sFc-HR/trimer elicited more robust and higher levels of neutralizing antibodies, with potent and broad neutralization activity against multiple SARS-CoV-2 variants. Notably, RBD-sFc-HR/trimer induced a durable immune response, significantly increasing the number of memory B cells and T cells. This study provides critical insights for designing vaccines that achieve potent and long-lasting immune responses against infectious diseases.

Structural basis of Epstein-Barr virus gp350 receptor recognition and neutralization.

Epstein-Barr virus (EBV) is an oncogenic virus associated with multiple lymphoid malignancies and autoimmune diseases. During infection in B cells, EBV uses its major glycoprotein gp350 to recognize the host receptor CR2, initiating viral attachment, a process that has lacked direct structural evidence for decades. In this study, we resolved the structure of the gp350-CR2 complex, elucidated their key interactions, and determined the site-specific N-glycosylation map of gp350. Our findings reveal that CR2 primarily binds to gp350 through an electrostatically complementary and glycan-free interface and that the diversity of key residues in CR2 across different species influences EBV host selectivity mediated by gp350. With the confirmed binding, we constructed a CR2-Fc antibody analog that targets the vulnerable site of gp350, demonstrating a potent neutralization effect against EBV infection in B cells. Our work provides essential structural insights into the mechanism of EBV infection and host tropism, suggesting a potential antiviral agent.

An allelic atlas of immunoglobulin heavy chain variable regions reveals antibody binding epitope preference resilient to SARS-CoV-2 mutation escape.

Although immunoglobulin (Ig) alleles play a pivotal role in the antibody response to pathogens, research to understand their role in the humoral immune response is still limited. We retrieved the germline sequences for the IGHV from the IMGT database to illustrate the amino acid polymorphism present within germline sequences of IGHV genes. We aassembled the sequences of IgM and IgD repertoire from 130 people to investigate the genetic variations in the population. A dataset comprising 10,643 SARS-CoV-2 spike-specific antibodies, obtained from COV-AbDab, was compiled to assess the impact of SARS-CoV-2 infection on allelic gene utilization. Binding affinity and neutralizing activity were determined using bio-layer interferometry and pseudovirus neutralization assays. Primary docking was performed using ZDOCK (3.0.2) to generate the initial conformation of the antigen-antibody complex, followed by simulations of the complete conformations using Rosetta SnugDock software. The original and simulated structural conformations were visualized and presented using ChimeraX (v1.5). We present an allelic atlas of immunoglobulin heavy chain (IgH) variable regions, illustrating the diversity of allelic variants across 33 IGHV family germline sequences by sequencing the IgH repertoire of in the population. Our comprehensive analysis of SARS-CoV-2 spike-specific antibodies revealed the preferential use of specific Ig alleles among these antibodies. We observed an association between Ig alleles and antibody binding epitopes. Different allelic genotypes binding to the same RBD epitope on the spike show different neutralizing potency and breadth. We found that antibodies carrying the IGHV1-69*02 allele tended to bind to the RBD E2.2 epitope. The antibodies carrying G50 and L55 amino acid residues exhibit potential enhancements in binding affinity and neutralizing potency to SARS-CoV-2 variants containing the L452R mutation on RBD, whereas R50 and F55 amino acid residues tend to have reduced binding affinity and neutralizing potency. IGHV2-5*02 antibodies using the D56 allele bind to the RBD D2 epitope with greater binding and neutralizing potency due to the interaction between D56 on HCDR2 and K444 on RBD of most Omicron subvariants. In contrast, IGHV2-5*01 antibodies using the N56 allele show increased binding resistance to the K444T mutation on RBD. This study provides valuable insights into humoral immune responses from the perspective of Ig alleles and population genetics. These findings underscore the importance of Ig alleles in vaccine design and therapeutic antibody development.

Evaluation of a surrogate virus neutralization assay for detecting neutralizing antibodies against SARS-CoV-2 in an African population.

The global resurgence of coronaviruses and the move to incorporate COVID-19 vaccines into the expanded program for immunization have warranted for a high-throughput and low-cost assay to measure and quantify mounted neutralizing antibodies as an indicator for protection against SARS-CoV-2. Hence, we evaluated the surrogate-virus-neutralization-assay (sVNT) as an alternative assay to the pseudo-virus neutralization assay (pVNT). The sVNT was used to measure neutralizing antibodies among 119 infected and/or vaccinated blood samples, against wild-type SARS-CoV-2 (WT) and the Omicron-variant with reference to the pVNT. Four different cut-offs were assessed for suitability in distinguishing neutralizers: the manufacturer (>30%), literature-based (>50%) and (>80%), and population-based (>27.69%). The obtained data was analyzed using "R" through its integrated development environments; JAMOV and R-Studio. Using the WT strain, only the population-based cut-off was able to differentiate neutralizers from non-neutralizers beyond chance, with an area under the curve (AUC) of 0.833 (95%CI, 0.505-1.0; P = .049). Applying the population-based cut-off, improved the sensitivity to 100% from 91.4% obtained from the manufacturer cut-off (P = .002). However, the specificity remained low (67%). The negative-predictive-value also improved to 100% vs 16.4% (P = .006), but there was no difference in the positive-predictive-value (99.1% vs 99.1%) (P = .340). When we used the Omicron-variant, the sVNT titers were not able to predict the neutralizers and non-neutralizers with reference to pVNT (AUC of 0.649) (P = .221). The sVNT assay is a potential alternative for screening individuals harboring potent neutralizing antibody with high sensitivity, although we recommend continuous improvement of the assay in line with the viral mutations. Further, we recommend that individual users establish a population-based cut-off while using the sVNT assay.

Enhanced Antibody Response to the Conformational Non-RBD Region via DNA Prime-Protein Boost Elicits Broad Cross-Neutralization Against SARS-CoV-2 Variants

Preventing immune escape of SARS-CoV-2 variants is crucial in vaccine development to ensure broad protection against the virus. Conformational epitopes beyond the RBD region are vital components of the spike protein but have received limited attention in the development of broadly protective SARS-CoV-2 vaccines. In this study, we used a DNA prime-protein boost regimen to evaluate the broad cross-neutralization potential of immune response targeting conformational non-RBD region against SARS-CoV-2 viruses in mice. Mice with enhanced antibody responses targeting conformational non-RBD region show better performance in cross-neutralization against the Wuhan-01, Delta, and Omicron subvariants. Via analyzing the distribution of conformational epitopes, and quantifying epitope-specific binding antibodies, we verified a positive correlation between the proportion of binding antibodies against the N-terminal domain (NTD) supersite (a conformational non-RBD epitope) and SARS-CoV-2 neutralization potency. The current work highlights the importance of high ratio of conformational non-RBD-specific binding antibodies in mediating viral cross-neutralization and provides new insight into overcoming the immune escape of SARS-CoV-2 variants.

Structural Immunology of SARS-CoV-2.

The SARS-CoV-2 spike (S) protein has undergone significant evolution, enhancing both receptor binding and immune evasion. In this review, we summarize ongoing efforts to develop antibodies targeting various epitopes of the S protein, focusing on their neutralization potency, breadth, and escape mechanisms. Antibodies targeting the receptor-binding site (RBS) typically exhibit high neutralizing potency but are frequently evaded by mutations in SARS-CoV-2 variants. In contrast, antibodies targeting conserved regions, such as the S2 stem helix and fusion peptide, exhibit broader reactivity but generally lower neutralization potency. However, several broadly neutralizing antibodies have demonstrated exceptional efficacy against emerging variants, including the latest omicron subvariants, underscoring the potential of targeting vulnerable sites such as RBS-A and RBS-D/CR3022. We also highlight public classes of antibodies targeting different sites on the S protein. The vulnerable sites targeted by public antibodies present opportunities for germline-targeting vaccine strategies. Overall, developing escape-resistant, potent antibodies and broadly effective vaccines remains crucial for combating future variants. This review emphasizes the importance of identifying key epitopes and utilizing antibody affinity maturation to inform future therapeutic and vaccine design.

Evaluation of cellular immune response in rabbits after exposure to cobra venom and purified toxin fraction

Snakebite by a cobra is considered neurotoxic as the cause of neuromuscular paralysis mediated by low molecular weight toxins, which are major toxin components of cobra. However, these toxins represent a problem in generating antibodies owing to their low immunogenicity. Developing complementary strategies to improve the antibody response could be a useful approach to creating better therapeutic antivenoms with higher neutralizing potencies. To develop simple immunization strategies for more potent antivenoms by studying the effects of combining crude cobra venom and toxin fraction in a complementary way. The evaluation of specific cell immunology and cytokine mediators for relevant immune responses will be measured in a rabbit model using four simple immunization strategies. Flow cytometry will be used to quantify the number of B and T cells, and qRT-PCR will be used to ascertain the cytokine genes expressed. B cells with anti-CD20 were seen on D14, and a booster dose was insufficient to maximize the antibodies. Conversely, anti-CD5 for T cells decreased periodically but remained stable. Using a mixture of crude cobra venom and its <10 kDa fraction, peak expression of pro-inflammatory cytokine genes was seen in D42 or D58, with a rise of 4 and 6 folds. Similarly, gene expression of pro-inflammatory cytokines was greater than that of anti-inflammatory cytokines (IL-4 and IL-10), which were up-regulated after D42. Thus, immunization with both the crude and its <10 kDa fraction of cobra venom seems to have synergistic effects that boost cytokines, activate the immune system, and cause lymphocyte differentiation.

Discovery of Nanosota-EB1 and -EB2 as Novel Nanobody Inhibitors Against Ebola Virus Infection.

The Ebola filovirus (EBOV) poses a serious threat to global health and national security. Nanobodies, a type of single-domain antibody, have demonstrated promising therapeutic potential. We identified two anti-EBOV nanobodies, Nanosota-EB1 and Nanosota-EB2, which specifically target the EBOV glycoprotein (GP). Cryo-EM and biochemical data revealed that Nanosota-EB1 binds to the glycan cap of GP1, preventing its protease cleavage, while Nanosota-EB2 binds to critical membrane-fusion elements in GP2, stabilizing it in the pre-fusion state. Nanosota-EB2 is a potent neutralizer of EBOV infection in vitro and offers excellent protection in a mouse model of EBOV challenge, while Nanosota-EB1 provides moderate neutralization and protection. Nanosota-EB1 and Nanosota-EB2 are the first nanobodies shown to inhibit authentic EBOV. Combined with our newly developed structure-guided in vitro evolution approach, they lay the foundation for nanobody-based therapies against EBOV and other viruses within the ebolavirus genus.

Interlayers geo-environmental assessment of phosphate waste rock for sustainable management practices.

Phosphate mines produce large quantities of waste rock. These waste rocks are mixed and managed on the surface as large unrestored piles, which makes them difficult to rehabilitate. They primarily comprise carbonates, clays, marls, and cherts (flints). In many cases, the unrestored mine sites, when exposed to normal climatic conditions, could frequently produce toxic environmental pollution, and significant ecological disruptions. This research aims to assess the phosphate waste rock's (PWR) geochemistry and environmental behavior upstream of the extraction process. For this purpose, different core drilling specimens and data were collected from different lithologies and depths in the interlayers of the Benguerir mine to forecast the environmental profile and determine the mobility of the analyzed chemical species. These samples were analyzed for their petrographical, chemical, and mineralogical compositions, static leaching tests, and semi-dynamic test. The mineralogy results showed that the PWR mainly consists of calcite, dolomite, apatite, and quartz, with minor phases such as clay minerals. Chemically, the PWRs are dominated by the following major oxides: CaO and MgO, followed by SiO2 and P2O5. Trace elements can be classified into three groups based on their concentrations: group of Sr, Zn and Cr (> 150ppm), group of Ba, V, Ni, Zr, Y, U, Cu, Cd, Co (10-150ppm), and group of trace elements with relatively low concentrations (< 10ppm): Rb, Pb, As, Mo, Se, Sc, Ga, Nb, Th, Hf, Sb and Cs. Environmentally, the pH of the leachates was neutral to alkaline (6 ± 0.6-9.3) for all the samples, which have a high neutralizing potential (38-991kg CaCO3/t). The release of major and trace elements in the leaching test remains below international standard limits. Consequently, the leaching test results confirm the non-hazardous nature of the PWR. Therefore, the studied PWR could be considered a natural raw material and can be used in various applications in different sectors, such as civil engineering, cement industry, phosphate recovery, and acid mine drainage treatment through neutralization.

Polyfunctionality and breadth of HIV-1 antibodies are associated with delayed disease progression.

HIV-1 infection leads to chronic disease requiring life-long treatment and therefore alternative therapeutics, a cure and/or a protective vaccine are needed. Antibody-mediated effector functions could have a role in the fight against HIV-1. However, the properties underlying the potential beneficial effects of antibodies during HIV-1 infection are poorly understood. To identify a specific profile of antibody features associated with delayed disease progression, we studied antibody polyfunctionality during untreated HIV-1 infection in the well-documented Amsterdam Cohort Studies. Serum samples were analyzed from untreated individuals with HIV-1 at approximately 6 months (n = 166) and 3 years (n = 382) post-seroconversion (post-SC). A Luminex antibody Fc array was used to profile 15 different Fc features for serum antibodies against 20 different HIV-1 envelope glycoprotein antigens and the resulting data was also compared with data on neutralization breadth. We found that high HIV-1 specific IgG1 levels and low IgG2 and IgG4 levels at 3 years post-SC were associated with delayed disease progression. Moreover, delayed disease progression was associated with a broad and polyfunctional antibody response. Specifically, the capacity to interact with all Fc γ receptors (FcγRs) and C1q, and in particular with FcγRIIa, correlated positively with delayed disease progression. There were strong correlations between antibody Fc features and neutralization breadth and several antibody features that were associated with delayed disease progression were also associated with the development of broad and potent antibody neutralization. In summary, we identified a strong association between broad, polyfunctional antibodies and delayed disease progression. These findings contribute new information for the fight against HIV-1, especially for new antibody-based therapy and cure strategies.

Discovery and engineering of the antibody response to a prominent skin commensal.

The ubiquitous skin colonist Staphylococcus epidermidis elicits a CD8+ T cell response pre-emptively, in the absence of an infection1. However, the scope and purpose of this anti-commensal immune program are not well defined, limiting our ability to harness it therapeutically. Here, we show that this colonist also induces a potent, durable, and specific antibody response that is conserved in humans and non-human primates. A series of S. epidermidis cell-wall mutants revealed that the cell surface protein Aap is a predominant target. By colonizing mice with a strain of S. epidermidis in which the parallel β-helix domain of Aap is replaced by tetanus toxin fragment C, we elicit a potent neutralizing antibody response that protects mice against a lethal challenge. A similar strain of S. epidermidis expressing an Aap-SpyCatcher chimera can be conjugated with recombinant immunogens; the resulting labeled commensal elicits high antibody titers under conditions of physiologic colonization, including a robust IgA response in the nasal and pulmonary mucosa. Thus, immunity to a common skin colonist involves a coordinated T and B cell response, the latter of which can be redirected against pathogens as a novel form of topical vaccination.

A novel monoclonal antibody against human thymic stromal lymphopoietin for the treatment of TSLP-mediated diseases.

Thymic stromal lymphopoietin (TSLP) is a master regulator of allergic inflammation against pathogens at barrier surfaces of the lung, skin, and gut. However, aberrant TSLP activity is implicated in various allergic, chronic inflammation and autoimmune diseases and cancers. Biologics drugs neutralizing excess TSLP activity represented by tezepelumab have been approved for severe asthma and are being evaluated for the treatments of other TSLP-mediated diseases. In this study, we discovered and characterized a novel humanized anti-TSLP antibody TAVO101 with high binding affinity to human TSLP, which blocks TSLP binding to its receptor complexes on cell surface. TAVO101 showed potent neutralization of TSLP activities in the TSLP-driven STAT5 reporter assay and cell proliferation assay. Results from ex vivo studies showed that TAVO101 neutralized TSLP-mediated CCL17 release from primary human CD1c+ dendritic cells and proliferation of activated CD4+ T cells. In addition, TAVO101 showed strong efficacy in both TSLP/OVA-induced asthma and imiquimod induced psoriasis models in hTSLP/hTSLPR double knock-in mice. We further conducted Fc engineering to optimize TAVO101 antibody with reduced affinity to Fcγ receptors and C1q protein but with increased affinity to FcRn receptor for half-life extension. By recognizing a different epitope, similarly potent neutralization of TSLP activities, and longer circulating half-life than tezepelumab, novel anti-TSLP antibody TAVO101 offers a potential best-in class therapeutics for various TSLP-mediated diseases.

Induction of Tier 2 HIV-Neutralizing IgA Antibodies in Rhesus Macaques Vaccinated with BG505.664 SOSIP.

A goal of mucosal human immunodeficiency virus type 1 (HIV-1) vaccines is to generate mucosal plasma cells producing polymeric IgA (pIgA)-neutralizing antibodies at sites of viral entry. However, vaccine immunogens capable of eliciting IgA neutralizing antibodies (nAbs) that recognize tier 2 viral isolates have not yet been identified. To determine if stabilized native-like HIV-1 envelope (Env) trimers could generate IgA nAbs, we purified total IgA and IgG from the banked sera of six rhesus macaques that had been found in a previous study to develop serum nAbs after subcutaneous immunization with BG505.664 SOSIP and 3M-052 adjuvant, which is a TLR7/8 agonist. The neutralization of autologous tier 2 BG505 T332N pseudovirus by the IgA and IgG preparations was measured using the TZM-bl assay. Anti-SOSIP binding antibodies (bAbs) were measured by ELISA. The IgG samples were found to have significantly greater levels of both nAb and bAb. However, after normalizing the nAb titer relative to the concentration of bAb, SOSIP-specific IgA purified from 2/6 animals was found to neutralize just as effectively as SOSIP-specific IgG, and in 3/6 animals, neutralization by the specific IgA was significantly greater. The more potent neutralization by IgA in these three animals was associated with a higher percentage of anti-SOSIP J chain-bound (polymeric) antibody. The parenteral vaccination of nonhuman primates with BG505.664 SOSIP generates HIV-1 tier 2 IgA nAbs in serum, including SOSIP-specific polymeric IgA, which appears to neutralize more efficiently than monomeric IgA or IgG. Mucosal delivery of this SOSIP or other stable Env trimers could generate locally synthesized polymeric IgA nAbs in mucosal tissues and secretions.

Development and application of an uncapped mRNA platform

Background A novel uncapped mRNA platform was developed. Methods Five lipid nanoparticle (LNP)-encapsulated mRNA constructs were made to evaluate several aspects of our platform, including transfection efficiency and durability in vitro and in vivo and the activation of humoral and cellular immunity in several animal models. The constructs were eGFP-mRNA-LNP (for enhanced green fluorescence mRNA), Fluc-mRNA-LNP (for firefly luciferase mRNA), SδT-mRNA-LNP (for Delta strain SARS-CoV-2 spike protein trimer mRNA), gDED-mRNA-LNP (for truncated glycoprotein D mRNA coding ectodomain from herpes simplex virus type 2 (HSV2)) and gDFR-mRNA-LNP (for truncated HSV2 glycoprotein D mRNA coding amino acids 1–400). Results Quantifiable target protein expression was achieved in vitro and in vivo with eGFP- and Fluc-mRNA-LNP. SδT-mRNA-LNP, gDED-mRNA-LNP and gDFR-mRNA-LNP induced both humoral and cellular immune responses comparable to those obtained by previously reported capped mRNA-LNP constructs. Notably, SδT-mRNA-LNP elicited neutralizing antibodies in hamsters against the Omicron and Delta strains. Additionally, gDED-mRNA-LNP and gDFR-mRNA-LNP induced potent neutralizing antibodies in rabbits and mice. The mRNA constructs with uridine triphosphate (UTP) outperformed those with N1-methylpseudouridine triphosphate (N1mψTP) in the induction of antibodies via SδT-mRNA-LNP. Conclusions Our uncapped, process-simplified and economical mRNA platform may have broad utility in vaccines and protein replacement drugs. KEY MESSAGES The mRNA platform described in our paper uses internal ribosome entry site (IRES) (Rapid, Amplified, Capless and Economical, RACE; Register as BH-RACE platform) instead of caps and uridine triphosphate (UTP) instead of N1-methylpseudouridine triphosphate (N1mψTP) to synthesize mRNA. Through the self-developed packaging instrument and lipid nanoparticle (LNP) delivery system, mRNA can be expressed in cells more efficiently, quickly and economically. Particularly exciting is that potent neutralizing antibodies against Delta and Omicron real viruses were induced with the new coronavirus S protein mRNA vaccine from the BH-RACE platform.