Potent Neutralizing Research Articles

Establishment of the First National Standard for Neutralizing Antibodies against SARS-CoV-2 XBB Variants.

Neutralizing antibodies (NtAbs) against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) are indicators of vaccine efficacy that enable immunity surveillance. However, the rapid mutation of SARS-CoV-2 variants prevents the timely establishment of standards required for effective XBB vaccine evaluation. Therefore, we prepared four candidate standards (No. 11, No. 44, No. 22, and No. 33) using plasma, purified immunoglobulin, and a broad-spectrum neutralizing monoclonal antibody. Collaborative calibration was conducted across nine Chinese laboratories using neutralization methods against 11 strains containing the XBB and BA.2.86 sublineages. This study demonstrated the reduced neutralization potency of the first International Standard antibodies to SARS-CoV-2 variants of concern against XBB variants. No. 44 displayed broad-spectrum neutralizing activity against XBB sublineages, effectively reduced interlaboratory variability for nearly all XBB variants, and effectively minimized the geometric mean titer (GMT) difference between the live and pseudotyped virus. No. 22 showed a broader spectrum and higher neutralizing activity against all strains but failed to reduce interlaboratory variability. Thus, No. 44 was approved as a National Standard for NtAbs against XBB variants, providing a unified NtAb measurement standard for XBB variants for the first time. Moreover, No. 22 was approved as a national reference reagent for NtAbs against SARS-CoV-2, offering a broad-spectrum activity reference for current and potentially emerging variants.

Establishment of replication-competent vesicular stomatitis virus recapitulating SADS-CoV entry.

Zoonotic coronaviruses pose a continuous threat to human health, with newly identified bat-borne viruses like swine acute diarrhea syndrome coronavirus (SADS-CoV) causing high mortality in piglets. In vitro studies indicate that SADS-CoV can infect cell lines from diverse species, including humans, highlighting its potential risk to human health. However, the lack of tools to study viral entry, along with the absence of vaccines or antiviral therapies, perpetuates this threat. To address this, we engineered an infectious molecular clone of Vesicular Stomatitis Virus (VSV), replacing its native glycoprotein (G) with SADS-CoV spike (S) and inserting a Venus reporter at the 3' leader region to generate a replication-competent rVSV-Venus-SADS S virus. Serial passages of rVSV-Venus-SADS S led to the identification of an 11-amino-acid truncation in the cytoplasmic tail of the S protein, which allowed more efficient viral propagation due to increased cell membrane anchoring of the S protein. The S protein was integrated into rVSV-Venus-SADS SΔ11 particles, susceptible to neutralization by sera from SADS-CoV S1 protein-immunized rabbits. Additionally, we found that TMPRSS2 promotes SADS-CoV spike-mediated cell entry. Furthermore, we assessed the serum-neutralizing ability of mice vaccinated with rVSV-Venus-SADS SΔ11 using a prime-boost immunization strategy, revealing effective neutralizing antibodies against SADS-CoV infection. In conclusion, we have developed a safe and practical tool for studying SADS-CoV entry and exploring the potential of a recombinant VSV-vectored SADS-CoV vaccine.IMPORTANCEZoonotic coronaviruses, like swine acute diarrhea syndrome coronavirus (SADS-CoV), pose a continual threat to human and animal health. To combat this, we engineered a safe and efficient tool by modifying the Vesicular Stomatitis Virus (VSV), creating a replication-competent rVSV-Venus-SADS S virus. Through serial passages, we optimized the virus for enhanced membrane anchoring, a key factor in viral propagation. This modified virus, rVSV-Venus-SADS SΔ11, proved susceptible to neutralization, opening avenues for potential vaccines. Additionally, our study revealed the role of TMPRSS2 in SADS-CoV entry. Mice vaccinated with rVSV-Venus-SADS SΔ11 developed potent neutralizing antibodies against SADS-CoV. In conclusion, our work presents a secure and practical tool for studying SADS-CoV entry and explores the promise of a recombinant VSV-vectored SADS-CoV vaccine.

A VHH single-domain platform enabling discovery and development of monospecific antibodies and modular neutralizing bispecifics against SARS-CoV-2 variants.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to evolve, escape coronavirus disease 2019 therapeutics and vaccines, and jeopardize public health. To combat SARS-CoV-2 antigenic escape, we developed a rapid, high-throughput pipeline to discover monospecific VHH antibodies and iteratively develop VHH-Fc-VHH bispecifics capable of neutralizing emerging SARS-CoV-2 variants. By panning VHH single-domain phage libraries against ancestral or beta spike proteins, we discovered high-affinity VHH antibodies with unique target epitopes. Combining two VHHs into a tetravalent bispecific construct conferred broad neutralization activity against multiple variants and was more resistant to antigenic escape than the monospecific antibody alone. Following the rise of the Omicron variant, a VHH in the original bispecific construct was replaced with another VHH discovered against the Omicron BA.1 receptor binding domain; the resulting bispecific exhibited neutralization against both BA.1 and BA.5 sublineage variants. A heavy chain-only tetravalent VHH-Fc-VHH bispecific platform derived from humanized synthetic libraries held a myriad of unique advantages: (i) synthetic preconstructed libraries minimized risk of liabilities and maximized discovery speed, (ii) VHH scaffolds allowed for a modular "plug-and-play" format that could be rapidly iterated upon as variants of concern arose, (iii) natural dimerization of single VHH-Fc-VHH polypeptides allowed for straightforward bispecific production and purification methods, and (iv) multivalent approaches enhanced avidity boosting effects and neutralization potency, and conferred more robust resistance to antigenic escape than monovalent approaches against specific variants. This iterative platform of rapid VHH discovery combined with modular bispecific design holds promise for long-term viral control efforts.

The SARS-CoV-2 neutralizing antibody response to SD1 and its evasion by BA.2.86

Under pressure from neutralising antibodies induced by vaccination or infection the SARS-CoV-2 spike gene has become a hotspot for evolutionary change, leading to the failure of all mAbs developed for clinical use. Most potent antibodies bind to the receptor binding domain which has become heavily mutated. Here we study responses to a conserved epitope in sub-domain-1 (SD1) of spike which have become more prominent because of mutational escape from antibodies directed to the receptor binding domain. Some SD1 reactive mAbs show potent and broad neutralization of SARS-CoV-2 variants. We structurally map the dominant SD1 epitope and provide a mechanism of action by blocking interaction with ACE2. Mutations in SD1 have not been sustained to date, but one, E554K, leads to escape from mAbs. This mutation has now emerged in several sublineages including BA.2.86, reflecting selection pressure on the virus exerted by the increasing prominence of the anti-SD1 response.

Anti–Interleukin-23 Autoantibodies in Adult-Onset Immunodeficiency

BackgroundAutoantibodies against interleukin-12 (anti–interleukin-12) are often identified in patients with thymoma, but opportunistic infections develop in only some of these patients. Interleukin-12 (with subunits p40 and p35) shares a common subunit with interleukin-23 (subunits p40 and p19). In a patient with disseminated Burkholderia gladioli infection, the identification of both anti–interleukin-23 and anti–interleukin-12 prompted further investigation.MethodsAmong the patients (most of whom had thymoma) who were known to have anti–interleukin-12, we screened for autoantibodies against interleukin-23 (anti–interleukin-23). To validate the potential role of anti–interleukin-23 with respect to opportunistic infection, we tested a second cohort of patients with thymoma as well as patients without either thymoma or known anti–interleukin-12 who had unusual infections.ResultsAmong 30 patients with anti–interleukin-12 who had severe mycobacterial, bacterial, or fungal infections, 15 (50%) also had autoantibodies that neutralized interleukin-23. The potency of such neutralization was correlated with the severity of these infections. The neutralizing activity of anti–interleukin-12 alone was not associated with infection. In the validation cohort of 91 patients with thymoma, the presence of anti–interleukin-23 was associated with infection status in 74 patients (81%). Overall, neutralizing anti–interleukin-23 was detected in 30 of 116 patients (26%) with thymoma and in 30 of 36 patients (83%) with disseminated, cerebral, or pulmonary infections. Anti–interleukin-23 was present in 6 of 32 patients (19%) with severe intracellular infections and in 2 of 16 patients (12%) with unusual intracranial infections, including Cladophialophora bantiana and Mycobacterium avium complex.ConclusionsAmong patients with a variety of mycobacterial, bacterial, or fungal infections, the presence of neutralizing anti–interleukin-23 was associated with severe, persistent opportunistic infections. (Funded by the National Institute of Allergy and Infectious Diseases and others.)

A Cocktail of Lipid Nanoparticle-mRNA Vaccines Broaden Immune Responses against β-Coronaviruses in a Murine Model.

Severe acute respiratory syndrome (SARS)-coronavirus (CoV), Middle Eastern respiratory syndrome (MERS)-CoV, and SARS-CoV-2 have seriously threatened human life in the 21st century. Emerging and re-emerging β-coronaviruses after the coronavirus disease 2019 (COVID-19) epidemic remain possible highly pathogenic agents that can endanger human health. Thus, pan-β-coronavirus vaccine strategies to combat the upcoming dangers are urgently needed. In this study, four LNP-mRNA vaccines, named O, D, S, and M, targeting the spike protein of SARS-CoV-2 Omicron, Delta, SARS-CoV, and MERS-CoV, respectively, were synthesized and characterized for purity and integrity. All four LNP-mRNAs induced effective cellular and humoral immune responses against the corresponding spike protein antigens in mice. Furthermore, LNP-mRNA S and D induced neutralizing antibodies against SARS-CoV and SARS-CoV-2, which failed to cross-react with MERS-CoV. Subsequent evaluation of sequential and cocktail immunizations with LNP-mRNA O, D, S, and M effectively elicited broad immunity against SARS-CoV-2 variants, SARS-CoV, and MERS-CoV. A direct comparison of the sequential with cocktail regimens indicated that the cocktail vaccination strategy induced more potent neutralizing antibodies and T-cell responses against heterotypic viruses as well as broader antibody activity against pan-β-coronaviruses. Overall, these results present a potential pan-β-coronavirus vaccine strategy for improved preparedness prior to future coronavirus threats.

Design of hepadnavirus core protein-based chimeric virus-like particles carrying epitopes from respiratory syncytial virus

Respiratory syncytial virus (RSV) is one of the most important pathogens causing respiratory tract infection in humans, especially in infants and the elderly. The identification and structural resolution of the potent neutralizing epitopes on RSV fusion (F) protein enable an “epitope-focused” vaccine design. However, the display of RSV F epitope II on the surface of the widely-used human hepatitis B virus core antigen (HBcAg) has failed to induce neutralizing antibody response in mice. Here, we used the hepadnavirus core protein (HcAg) from different mammalian hosts as scaffolds to construct chimeric virus-like particles (VLPs) presenting the RSV F epitope II. Mouse immunization showed that different HcAg-based chimeric VLPs elicited significantly different neutralizing antibody responses, among which the HcAg derived from roundleaf bat (RBHcAg) is the most immunogenic. Furthermore, RBHcAg was used as the scaffold platform to present multiple RSV F epitopes, and the immunogenicity was further improved in comparison to that displaying a single epitope II. The designed RBHcAg-based multiple-epitope-presenting VLP formulated with MF59-like adjuvant elicited a potent and balanced Th1/Th2 immune response, and offered substantial protection in mice against the challenge of live RSV A2 virus. The designed chimeric VLPs may serve as the potential starting point for developing epitope-focused vaccines against RSV. Our study also demonstrated that RBHcAg is an effective VLP carrier for presenting foreign epitopes, providing a promising platform for epitope-focused vaccine design.

Functional dissection of the spike glycoprotein S1 subunit and identification of cellular cofactors for regulation of swine acute diarrhea syndrome coronavirus entry.

Swine acute diarrhea syndrome coronavirus (SADS-CoV) is a novel porcine enteric coronavirus, and the broad interspecies infection of SADS-CoV poses a potential threat to human health. This study provides experimental evidence to dissect the roles of distinct domains within the SADS-CoV spike S1 subunit in cellular entry. Specifically, we expressed the S1 and its subdomains, S1A and S1B. Cell binding and invasion inhibition assays revealed a preference for the S1B subdomain in binding to the receptors on the cell surface, and this unknown receptor is not utilized by the porcine epidemic diarrhea virus. Nanoparticle display demonstrated hemagglutination of erythrocytes from pigs, humans, and mice, linking the S1A subdomain to the binding of sialic acid (Sia) involved in virus attachment. We successfully rescued GFP-labeled SADS-CoV (rSADS-GFP) from a recombinant cDNA clone to track viral infection. Antisera raised against S1, S1A, or S1B contained highly potent neutralizing antibodies, with anti-S1B showing better efficiency in neutralizing rSADS-GFP infection compared to anti-S1A. Furthermore, depletion of heparan sulfate (HS) by heparinase treatment or pre-incubation of rSADS-GFP with HS or constituent monosaccharides could inhibit SADS-CoV entry. Finally, we demonstrated that active furin cleavage of S glycoprotein and the presence of type II transmembrane serine protease (TMPRSS2) are essential for SADS-CoV infection. These combined observations suggest that the wide cell tropism of SADS-CoV may be related to the distribution of Sia or HS on the cell surface, whereas the S1B contains the main protein receptor binding site. Specific host proteases also play important roles in facilitating SADS-CoV entry.IMPORTANCESwine acute diarrhea syndrome coronavirus (SADS-CoV) is a novel pathogen infecting piglet, and its unique genetic evolution characteristics and broad species tropism suggest the potential for cross-species transmission. The virus enters cells through its spike (S) glycoprotein. In this study, we identify the receptor binding domain on the C-terminal part of the S1 subunit (S1B) of SADS-CoV, whereas the sugar-binding domain located at the S1 N-terminal part of S1 (S1A). Sialic acid, heparan sulfate, and specific host proteases play essential roles in viral attachment and entry. The dissection of SADS-CoV S1 subunit's functional domains and identification of cellular entry cofactors will help to explore the receptors used by SADS-CoV, which may contribute to exploring the mechanisms behind cross-species transmission and host tropism.

An in vivo BSL-2 model for henipavirus infection based on bioluminescence imaging of recombinant Cedar virus replication in mice.

Henipaviruses are enveloped single-stranded, negative-sense RNA viruses of the paramyxovirus family. Two henipaviruses, Nipah virus and Hendra virus, cause a systemic respiratory and/or neurological disease in humans and ten additional species of mammals, with a high fatality rate. Because of their highly pathogenic nature, Nipah virus and Hendra virus are categorized as BSL-4 pathogens, which limits the number and scope of translational research studies on these important human pathogens. To begin to address this limitation, we are developing a BSL-2 model of authentic henipavirus infection in mice, using the non-pathogenic henipavirus, Cedar virus. Notably, wild-type mice are highly resistant to Hendra virus and Nipah virus infection. However, previous work has shown that mice lacking expression of the type I interferon receptor (IFNAR-KO mice) are susceptible to both viruses. Here, we show that luciferase-expressing recombinant Cedar virus (rCedV-luc) is also able to replicate and establish a transient infection in IFNAR-KO mice, but not in wild-type mice. Using longitudinal bioluminescence imaging (BLI) of luciferase expression, we detected rCedV-luc replication as early as 10 h post-infection. Viral replication peaks between days 1 and 3 post-infection, and declines to levels undetectable by BLI by 7 days post-infection. Immunohistochemistry is consistent with viral infection and replication in endothelial cells and other non-immune cell types within tissue parenchyma. Serology analyses demonstrate significant IgG responses to the Cedar virus surface glycoprotein with potent neutralizing activity in IFNAR-KO mice, whereas antibody responses in wild-type animals were non-significant. Overall, these data suggest that rCedV-luc infection of IFNAR-KO mice represents a viable platform for the study of in vivo henipavirus replication, anti-henipavirus host responses and henipavirus-directed therapeutics.

Immunochemical Recognition of Bothrops rhombeatus Venom by Two Polyvalent Antivenoms.

The protein profile of Bothrops rhombeatus venom was compared to Bothrops asper and Bothrops atrox, and the effectiveness of antivenoms from the National Institute of Health of Colombia (INS) and Antivipmyn-Tri (AVP-T) of Mexico were analyzed. Protein profiles were studied with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and reverse-phase high-performance liquid chromatography (RP-HPLC). The neutralizing potency and the level of immunochemical recognition of the antivenoms to the venoms were determined using Western blot, affinity chromatography, and enzyme-linked immunosorbent assay (ELISA). Bands of phospholipase A2 (PLA2), metalloproteinases (svMPs) I, II, and III as well as serine proteinases (SPs) in the venom of B. rhombeatus were recognized by SDS-PAGE. With Western blot, both antivenoms showed immunochemical recognition towards PLA2 and svMP. INS showed 94% binding to B. rhombeatus venom and 92% to B. asper while AVP-T showed 90.4% binding to B. rhombeatus venom and 96.6% to B. asper. Both antivenoms showed binding to PLA2 and svMP, with greater specificity of AVP-T towards B. rhombeatus. Antivenom neutralizing capacity was calculated by species and mL of antivenom, finding the following for INS: B. asper 6.6 mgV/mL, B. atrox 5.5 mgV/mL, and B. rhombeatus 1.3 mgV/mL. Meanwhile, for AVP-T, the following neutralizing capacities were found: B. asper 2.7 mgV/mL, B. atrox 2.1 mgV/mL, and B. rhombeatus 1.4 mgV/mL. These results show that both antivenoms presented similarity between calculated neutralizing capacities in our trial, reported in a product summary for the public for the B. asper species; however, this does not apply to the other species tested in this trial.

In vivo affinity maturation of mouse B cells reprogrammed to express human antibodies.

Mice adoptively transferred with mouse B cells edited via CRISPR to express human antibody variable chains could help evaluate candidate vaccines and develop better antibody therapies. However, current editing strategies disrupt the heavy-chain locus, resulting in inefficient somatic hypermutation without functional affinity maturation. Here we show that these key B-cell functions can be preserved by directly and simultaneously replacing recombined mouse heavy and kappa chains with those of human antibodies, using a single Cas12a-mediated cut at each locus and 5' homology arms complementary to distal V segments. Cells edited in this way to express the human immunodeficiency virus type 1 (HIV-1) broadly neutralizing antibody 10-1074 or VRC26.25-y robustly hypermutated and generated potent neutralizing plasma in vaccinated mice. The 10-1074 variants isolated from the mice neutralized a global panel of HIV-1 isolates more efficiently than wild-type 10-1074 while maintaining its low polyreactivity and long half-life. We also used the approach to improve the potency of anti-SARS-CoV-2 antibodies against recent Omicron strains. In vivo affinity maturation of B cells edited at their native loci may facilitate the development of broad, potent and bioavailable antibodies.

Broad Epitope Coverage of Therapeutic Multi-Antibody Combinations Targeting SARS-CoV-2 Boosts In Vivo Protection and Neutralization Potency to Corner an Immune-Evading Virus.

Therapeutic antibodies (Abs) which act on a broader range of epitopes may provide more durable protection against the genetic drift of a target, typical of viruses or tumors. When these Abs exist concurrently on the targeted antigen, several mechanisms of action (MoAs) can be engaged, boosting therapeutic potency. This study selected combinations of four and five Abs with non- or partially overlapping epitopes to the SARS-CoV-2 spike glycoprotein, on or outside the crucial receptor binding domain (RBD), to offer resilience to emerging variants and trigger multiple MoAs. The combinations were derived from a pool of unique-sequence scFv Ab fragments retrieved from two SARS-CoV-2-naïve human phage display libraries. Following recombinant expression to full-length human IgG1 candidates, a biolayer interferometric analysis mapped epitopes to bins and confirmed that up to four Abs from across the bins can exist simultaneously on the spike glycoprotein trimer. Not all the bins of Abs interfered with the spike protein binding to angiotensin converting enzyme 2 (ACE2) in competitive binding assays, nor neutralized the pseudovirus or authentic virus in vitro, but when combined in vivo, their inclusion resulted in a much stronger viral clearance in the lungs of intranasally challenged hamsters, compared to that of those treated with mono ACE2 blockers. In addition, the Ab mixtures activated in vitro reporter cells expressing Fc-gamma receptors (FcγRs) involved in antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis (ADCP). The best four-Ab combination neutralized seventeen variants of concern from Wuhan-Hu1 to Omicron BA.4/BA.5 in vitro.

Comparative Immunogenicity and Neutralization Potency of Four Approved COVID-19 Vaccines in BALB/c Mice.

Since the outbreak of the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), several vaccine candidates have been developed within a short period of time. Although the potency of these vaccines was evaluated individually, their comparative potency was not comprehensively evaluated. To compare the immunogenicity and neutralization efficacy of four approved COVID-19 vaccines in Iran, including: PastoCovac Plus, Sinopharm, SpikoGen, and Noora in BALB/c mice. Different groups of female BALB/c mice were vaccinated with three doses of each vaccine. The serum levels of antibodies against the viral receptor binding domain (anti-RBD) and spike (anti-spike) protein as well as the vaccine formulation (anti-vaccine) were evaluated using enzyme-linked immunosorbent assay (ELISA). The neutralization efficacy of these four vaccines was assessed through four neutralization assays: conventional virus neutralization test (cVNT), pseudotype virus neutralization test (pVNT), surrogate virus neutralization test (sVNT), and inhibition flow cytometry. All four vaccines induced seroconversion in vaccinated animals. All vaccines successfully induced high levels of anti-vaccine antibody; however, PastoCovac Plus and Sinopharm vaccines induced significantly higher levels of anti-RBD antibody titer compared to Noora and SpikoGen. Moreover, the results of the antibody response were corroborated by the virus neutralization tests, which revealed very weak neutralization potency by Noora and SpikoGen in all tests. Our results indicate significant immunogenicity and neutralization efficacy induced by PastoCovac Plus and Sinopharm, but not by Noora and SpikoGen. This suggests the need for additional comparative assessment of the potency and efficacy of these four vaccines in vaccinated subjects.

Virus-like particles of louping ill virus elicit potent neutralizing antibodies targeting multimers of viral envelope protein

Louping ill virus (LIV) is a tick-borne flavivirus that predominantly causes disease in livestock, especially sheep in the British Isles. A preventive vaccine, previously approved for veterinary use but now discontinued, was based on an inactivated whole virion that likely provided protection by induction of neutralizing antibodies recognizing the viral envelope (E) protein. A major disadvantage of the inactivated vaccine was the need for high containment facilities for the propagation of infectious virus, as mandated by the hazard group 3 status of the virus. This study aimed to develop high-efficacy non-infectious protein-based vaccine candidates. Specifically, soluble envelope protein (sE), and virus-like particles (VLPs), comprised of the precursor of membrane and envelope proteins, were generated, characterized, and studied for their immunogenicity in mice. Results showed that the VLPs induced more potent virus neutralizing response compared to sE, even though the total anti-envelope IgG content induced by the two antigens was similar. Depletion of anti-monomeric E protein antibodies from mouse immune sera suggested that the neutralizing antibodies elicited by the VLPs targeted epitopes spanning the highly organized structure of multimer of the E protein, whereas the antibody response induced by sE focused on E monomers. Thus, our results indicate that VLPs represent a promising LIV vaccine candidate.

PGM-CSF as an adjuvant in DNA vaccination against SARS-CoV-2

The emergence of SARS-CoV-2 presents a significant global public health dilemma. Vaccination has long been recognized as the most effective means of preventing the spread of infectious diseases. DNA vaccines have attracted attention due to their safety profile, cost-effectiveness, and ease of production. This study aims to assess the efficacy of plasmid-encoding GM-CSF (pGM-CSF) as an adjuvant to augment the specific humoral and cellular immune response elicited by DNA vaccines based on the receptor-binding domain (RBD) antigen. Compared to the use of plasmid-encoded RBD (pRBD) alone, mice that were immunized with a combination of pRBD and pGM-CSF exhibited significantly elevated levels of RBD-specific antibody titers in serum, BALF, and nasal wash. Furthermore, these mice generated more potent neutralization antibodies against both the wild-type and Omicron pseudovirus, as well as the ancestral virus. In addition, pGM-CSF enhanced pRBD-induced CD4+ and CD8+ T cell responses and promoted central memory T cells storage in the spleen. At the same time, tissue-resident memory T (Trm) cells in the lung also increased significantly, and higher levels of specific responses were maintained 60 days post the final immunization. pGM-CSF may play an adjuvant role by promoting antigen expression, immune cells recruitment and GC B cell responses. In conclusion, pGM-CSF may be an effective adjuvant candidate for the DNA vaccines against SARS-CoV-2.

Sixth monovalent XBB.1.5 vaccine elicits robust immune response against emerging SARS-CoV-2 variants in heart transplant recipients

Continued circulation of SARS-CoV-2 has driven the selection of variants with improved ability to escape pre-existing vaccine-induced responses, posing a persistent threat to heart transplant recipients (HTRs). The immunogenicity and safety of the updated XBB.1.5-containing monovalent vaccines are unknown. We prospectively enrolled 52 HTRs who had previously received a 5-dose ancestral-derived monovalent and bivalent mRNA vaccination schedule to receive the monovalent XBB.1.5 vaccine. Immunogenicity was evaluated using live virus micro-neutralization assays. The XBB.1.5 monovalent vaccine elicited potent and diverse neutralizing responses and broadened the reactivity spectrum to encompass newer strains, with the highest increase in neutralization activity being more pronounced against XBB.1.5 (15.8-fold) and JN.1 (13.3-fold) than against BA.5 (6.7-fold) and WT (4-fold). Notably, XBB.1.5 and JN.1 were resistant to neutralization by pre-vaccination sera. There were no safety concerns. Our findings support updating of COVID-19 vaccines to match antigenically divergent variants and exclude ancestral spike-antigen to protect HTRs.

Fast-Track Discovery of SARS-CoV-2-Neutralizing Antibodies from Human B Cells by Direct Functional Screening.

As the COVID-19 pandemic revealed, rapid development of vaccines and therapeutic antibodies are crucial to guarantee a quick return to the status quo of society. In early 2020, we deployed our droplet microfluidic single-cell-based platform DROPZYLLA® for the generation of cognate antibody repertoires of convalescent COVID-19 donors. Discovery of SARS-CoV-2-specific antibodies was performed upon display of antibodies on the surface of HEK293T cells by antigen-specific sorting using binding to the SARS-CoV-2 spike and absence of binding to huACE2 as the sort criteria. This efficiently yielded antibodies within 3-6 weeks, of which up to 100% were neutralizing. One of these, MTX-COVAB, displaying low picomolar neutralization IC50 of SARS-CoV-2 and with a neutralization potency on par with the Regeneron antibodies, was selected for GMP manufacturing and clinical development in June 2020. MTX-COVAB showed strong efficacy in vivo and neutralized all identified clinically relevant variants of SARS-CoV-2 at the time of its selection. MTX-COVAB completed GMP manufacturing by the end of 2020, but clinical development was stopped when the Omicron variant emerged, a variant that proved to be detrimental to all monoclonal antibodies already approved. The present study describes the capabilities of the DROPZYLLA® platform to identify antibodies of high virus-neutralizing capacity rapidly and directly.

Immunogenicity phase II study evaluating booster capacity of nonadjuvanted AKS-452 SARS-Cov-2 RBD Fc vaccine.

AKS-452, a subunit vaccine comprising an Fc fusion of the ancestral wild-type (WT) SARS-CoV-2 virus spike protein receptor binding domain (SP/RBD), was evaluated without adjuvant in a single cohort, non-randomized, open-labelled phase II study (NCT05124483) at a single site in The Netherlands for safety and immunogenicity. A single 90 µg subcutaneous booster dose of AKS-452 was administered to 71 adults previously primed with a registered mRNA- or adenovirus-based vaccine and evaluated for 273 days. All AEs were mild and no SAEs were attributable to AKS-452. While all subjects showed pre-existing SP/RBD binding and ACE2-inhibitory IgG titers, 60-68% responded to AKS-452 via ≥2-fold increase from days 28 to 90 and progressively decreased back to baseline by day 180 (days 28 and 90 mean fold-increases, 14.7 ± 6.3 and 8.0 ± 2.2). Similar response kinetics against RBD mutant proteins (including omicrons) were observed but with slightly reduced titers relative to WT. There was an expected strong inverse correlation between day-0 titers and the fold-increase in titers at day 28. AKS-452 enhanced neutralization potency against live virus, consistent with IgG titers. Nucleocapsid protein (Np) titers suggested infection occurred in 66% (46 of 70) of subjects, in which only 20 reported mild symptomatic COVID-19. These favorable safety and immunogenicity profiles support booster evaluation in a planned phase III universal booster study of this room-temperature stable vaccine that can be rapidly and inexpensively manufactured to serve vaccination at a global scale without the need of a complex distribution or cold chain.

Bovine ultralong CDR-H3 derived knob paratopes elicit potent TNF-α neutralization and enable the generation of novel adalimumab-based antibody architectures with augmented features.

In this work we have generated cattle-derived chimeric ultralong CDR-H3 antibodies targeting tumor necrosis factor α (TNF-α) via immunization and yeast surface display. We identified one particular ultralong CDR-H3 paratope that potently neutralized TNF-α. Interestingly, grafting of the knob architecture onto a peripheral loop of the CH3 domain of the Fc part of an IgG1 resulted in the generation of a TNF-α neutralizing Fc (Fcknob) that did not show any potency loss compared with the parental chimeric IgG format. Eventually, grafting this knob onto the CH3 region of adalimumab enabled the engineering of a novel TNF-α targeting antibody architecture displaying augmented TNF-α inhibition.

B cell focused transient immune suppression protocol for efficient AAV readministration to the liver

Adeno associated virus (AAV) vectors are utilized for correcting multiple genetic disorders. Although the goal is to achieve life-long correction with a single vector administration, the ability to redose would enable extension of therapy in cases where initial gene transfer is insufficient to achieve a lasting cure, episomal vector forms are lost in growing organs of pediatric patients, or transgene expression is diminished over time. However, AAV typically induces potent and long-lasting neutralizing antibodies (NAb) against capsid that prevents re-administration. To prevent NAb formation in hepatic AAV8 gene transfer, we developed a transient B cell targeting protocol using a combination of monoclonal antibody therapy against CD20 (for B cell depletion) and BAFF (to slow B cell repopulation). Initiation of immunosuppression prior to (rather than at the time of) vector administration and prolonged anti-BAFF treatment prevented immune responses against the transgene product and abrogated prolonged IgM formation. As a result, vector re-administration after immune reconstitution was highly effective. Interestingly, re-administration before the immune system had fully recovered achieved further elevated levels of transgene expression. Finally, this immunosuppression protocol reduced Ig mediated AAV uptake by immune cell types with implications to reduce the risk of immunotoxicities in human gene therapy with AAV.