Determination of the Effects of Prenatal Remdesivir Exposure on Placenta Morphology, Placental IgG Transfer, and Fetal Growth in a Humanized Mouse Model

Remdesivir (RDV) is an antiviral medication used most recently for the treatment of COVID-19. Although no adverse effects were observed on perinatal parameters in reproductive and development toxicology studies at doses up to four-fold clinical area under the curve (AUC) exposures, some researchers have reported that therapeutic levels of RDV may impair early embryogenesis, as observed by in vitro studies. In addition, the influence of prenatal RDV exposure on maternal IgG transfer in the placenta is still unknown. Administration of RDV in pregnant humanized mouse model (Tg32), which expresses the human Fc gamma receptor and transporter (FCGRT) gene, was used to further evaluate potential effects on IgG transfer and concurrent perinatal endpoints. Animals were dosed daily from gestational days (GDs) 10- 14 with 25 mg/kg RDV (GS-5734) via intravenous injection (n=3-5 per group). Concurrent vehicle control animals were dosed intravenously with 12% sulfobutyl ether- beta-cyclodextrin in water (pH3.5;NaOH/HCl). All animals were administered 2 mg/kg human IgG via intravenous injection on GD 14. Placentae and fetuses were collected from dams on GD 14, 15, 16, and 18 and evaluated using histopathology and qPCR for inflammation markers. No abnormal morphologies (necrosis/apoptosis) of placentae were observed between the concurrent control and RDVdosed groups. Additionally, no differences in maternal body weights were observed. There were no statistically significant differences in placenta weights. There were no statistically significant changes in pregnancy parameters (implantation sites and dead fetuses/litter) and fetal weights between the RDV-dosed group and concurrent controls at GD 14, 15, 16, and 18. No changes were observed in transcript levels of inflammation markers in the RDV-dosed group when compared to the concurrent control group. There was a slightly lower ratio of fetal IgG level to maternal IgG levels in the RDV-dosed group;however, no statistically significant differences were observed between the RDV-dosed group and concurrent controls on GD 14, 15, 16, and 18. Our results suggest that a daily dose of 25 mg/kg RDV on GDs 10-14 in humanized mice did not cause adverse effects on placenta and fetal development. (Funded by the Perinatal Health Center of Excellence: E0300201.).

Coronavac inactivated vaccine triggers durable, cross-reactive Fc-mediated phagocytosis activities.

Although humoral responses elicited by infection or vaccine lost the ability to prevent transmission against Omicron, vaccine-induced antibodies may still contribute to disease attenuation through Fc-mediated effector functions. However, Fc effector function elicited by CoronaVac, as the most widely supplied inactivated vaccine globally, has not been characterized. For the first time, our study depicted Fc-mediated phagocytosis activity induced by CoronaVac, including antibody-dependent cellular phagocytosis (ADCP) and antibody-dependent neutrophil phagocytosis (ADNP) activities, and further compared with that from convalescent individuals and CoronaVac recipients with subsequent breakthrough infections. We showed that 2-dose of CoronaVac effectively induced both ADCP and ADNP, but was substantially lower compared to infection, whereas the booster dose further augmented ADCP and ADNP responses, and remained detectable for 52 weeks. Among CoronaVac recipients, ADCP and ADNP responses also demonstrated cross-reactivity against Omicron subvariants, and breakthrough infection could enhance the phagocytic response. Meanwhile, serum samples from vaccinees, convalescent individuals with wildtype infection, BA.2 and BA.5 breakthrough infection demonstrated differential cross-reactive ADCP and ADNP responses against Omicron subvariants, suggesting the different subvariants of spike antigen exposure might alter the cross-reactivity of Fc effector function. Further, ADCP and ADNP responses were strongly correlated with Spike-specific IgG responses and neutralizing activities, indicating coordinated neutralization activity, ADCP and ADNP responses triggered by CoronaVac. Of note, the ADCP and ADNP responses were more durable and cross-reactive than corresponding Spike-specific IgG titers and neutralizing activities. Our study has important implications for optimal boosting vaccine strategies that may induce potent and broad Fc-mediated phagocytic activities.

A Dual-Approach Strategy to Optimize the Safety and Efficacy of Anti-Zika Virus Monoclonal Antibody Therapeutics.

Antibody-dependent enhancement of infection (ADE) is clinically relevant to Dengue virus (DENV) infection and poses a major risk to the application of monoclonal antibody (mAb)-based therapeutics against related flaviviruses such as the Zika virus (ZIKV). Here, we tested a two-tier approach for selecting non-cross-reactive mAbs combined with modulating Fc glycosylation as a strategy to doubly secure the elimination of ADE while preserving Fc effector functions. To this end, we selected a ZIKV-specific mAb (ZV54) and generated three ZV54 variants using Chinese hamster ovary cells and wild-type (WT) and glycoengineered ΔXF Nicotiana benthamiana plants as production hosts (ZV54CHO, ZV54WT, and ZV54ΔXF). The three ZV54 variants shared an identical polypeptide backbone, but each exhibited a distinct Fc N-glycosylation profile. All three ZV54 variants showed similar neutralization potency against ZIKV but no ADE activity for DENV infection, validating the importance of selecting the virus/serotype-specific mAbs for avoiding ADE by related flaviviruses. For ZIKV infection, however, ZV54CHO and ZV54ΔXF showed significant ADE activity while ZV54WT completely forwent ADE, suggesting that Fc glycan modulation may yield mAb glycoforms that abrogate ADE even for homologous viruses. In contrast to the current strategies for Fc mutations that abrogate all effector functions along with ADE, our approach allowed the preservation of effector functions as all ZV54 glycovariants retained antibody-dependent cellular cytotoxicity (ADCC) against the ZIKV-infected cells. Furthermore, the ADE-free ZV54WT demonstrated in vivo efficacy in a ZIKV-infection mouse model. Collectively, our study provides further support for the hypothesis that antibody-viral surface antigen and Fc-mediated host cell interactions are both prerequisites for ADE, and that a dual-approach strategy, as shown herein, contributes to the development of highly safe and efficacious anti-ZIKV mAb therapeutics. Our findings may be impactful to other ADE-prone viruses, including SARS-CoV-2.

Multifaceted roles of Fcγ receptors in COVID-19 and vaccine responses.

Recent data have revealed various effector functions of FcγRs in immune responses against challenges with SARS-CoV-2 virus. FcγRs act as a bridge between antibody specificity and effector cells. In many cases, IgG/FcγR interactions generate cell-mediated immune protection from infection via ADCP or ADCC. These responses are beneficial, as they may participate in virus elimination and persist longer than neutralizing anti-Spike antibodies. In contrast, these interactions may sometimes prove beneficial to the virus by enhancing viral uptake into phagocytic cells via ADE and causing excessive inflammation. Here, we summarize key features of FcγRs, discuss effector functions, clinical relevance, and factors influencing FcγR-mediated immune responses in COVID-19 and vaccine responses, and consider IVIg and kinase inhibitors for targeting FcγRs signaling in COVID-19.

Complement Activation by an Anti-Dengue/Zika Antibody with Impaired Fcγ Receptor Binding Provides Strong Efficacy and Abrogates Risk of Antibody-Dependent Enhancement.

To combat infectious diseases, vaccines are considered the best prophylactic strategy for a wide range of the population, but even when vaccines are effective, the administration of therapeutic antibodies against viruses could provide further treatment options, particularly for vulnerable groups whose immunity against the viruses is compromised. Therapeutic antibodies against dengue are ideally engineered to abrogate binding to Fcγ receptors (FcγRs), which can induce antibody-dependent enhancement (ADE). However, the Fc effector functions of neutralizing antibodies against SARS-CoV-2 have recently been reported to improve post-exposure therapy, while they are dispensable when administered as prophylaxis. Hence, in this report, we investigated the influence of Fc engineering on anti-virus efficacy using the anti-dengue/Zika human antibody SIgN-3C and found it affected the viremia clearance efficacy against dengue in a mouse model. Furthermore, we demonstrated that complement activation through antibody binding to C1q could play a role in anti-dengue efficacy. We also generated a novel Fc variant, which displayed the ability for complement activation but showed very low FcγR binding and an undetectable level of the risk of ADE in a cell-based assay. This Fc engineering approach could make effective and safe anti-virus antibodies against dengue, Zika and other viruses.

Impact of Covid-19 and Host Factors on the Humoral Immune Repertoire in Treated Hiv

Background: People with HIV (PWH) on antiretroviral therapy (ART) appear to be at higher risk for worse COVID-19 outcomes, but the underlying mechanisms-including effects of COVID-19 and host factors on the broader humoral immune repertoire-are poorly understood. Method(s): REPRIEVE enrolled a global cohort of ART-treated PWH ages 40-75. COVID+ was defined by positive receptor binding domain IgG or IgA from annual visits 5/2020-2/2021. Antibody isotype, subclass, and Fc receptor Luminex arrays to SARS-CoV-2, CMV, EBV, HSV, HIV, influenza, pneumococcus, and RSV were assessed. Report of COVID diagnosis (collected every 4 months) was defined as mild, moderate, or severe (asymptomatic if no clinical diagnosis but IgG/ IgA+). FDR-corrected regression was used to assess effects of 1) COVID+ on non- SARS-CoV-2 repertoire in full cohort and 2) host factors on non-SARS-CoV-2 and SARS-CoV-2 repertoire in COVID- and COVID+ cohorts, respectively, adjusted for age, sex, region, nadir CD4, and HIV VL at entry. Result(s): Of 2,464 unvaccinated participants, 283 (11%) were COVID+;260 (92%) were asymptomatic. Median age was 53, 35% were women, 50% had nadir CD4 < 200, median current CD4 was 649, and 97% had HIV VL < 400. In the full cohort, COVID+ was associated with higher CMV PP65 IgG3 and FcgammaRIIA (P< 0.05);COVID severity was not associated with the non-SARS-CoV-2 repertoire. Among COVID-, older age, female sex, and lower nadir CD4 were associated with higher EBV and CMV responses;IgG1 levels were higher in women for all non-SARS-CoV-2 antigens assessed (P< 0.05). Among COVID+, higher BMI was associated with amplified SARS-CoV-2 IgG, IgA, IgM, and FcgammaRIIA responses (P< 0.05). Lower nadir CD4 was associated with a SARSCoV- 2 repertoire shift toward IgM and FcgammaRIIB (P< 0.05). Age and sex were not associated with SARS-CoV-2-related repertoire changes in COVID+. Conclusion(s): Our analysis presents a comprehensive view of host factors associated with the humoral immune repertoire among a global cohort of ART-treated PWH. COVID's association with higher CMV responses may suggest increased susceptibility to or a consequence of persistent inflammation after infection. The striking amplification of SARS-CoV-2 responses with higher BMI suggests an excessive inflammatory response. Lower nadir CD4 was related to uncontrolled extra-follicular and inhibitory SARS-CoV-2 responses, which are unlikely to be protective. These findings may suggest mechanisms underlying factors associated with worse COVID-19 outcomes among PWH. (Figure Presented).

Fc RECEPTOR-MEDIATED INFECTION OF MYELOID CELLS BY SARS-CoV-2

Background: The role of myeloid cells in the pathogenesis of SARS-CoV-2 is well established, in particular as drivers of cytokine production and systemic inflammation characteristic of severe COVID-19. However, the potential for myeloid cells to act as bona fide targets of productive SARS-CoV-2 infection remains unclear. Method(s): Using anti-SARS-CoV-2 mAbs with a range of neutralisation potencies and binding specificities, we performed a detailed assessment of mAb-mediated infection of monocytes/macrophages. THP-1 cells were used as a model system, with results confirmed in primary macrophages. Result(s): Infection of THP-1 cells was seen via mAbs targeting the spike RBD, but not with those targeting the NTD or S2 subunit. mAbs with the most consistent potential to mediate infection targeted a conserved region of the RBD (group 1/class IV). No infection was seen with the same quantity of virus but in the absence of antibody, and pre-treating the cells with FcgammaRI and -II blocking antibodies inhibited infection. Thus, antibody-FcR interactions are able to expand the tropism of SARS-CoV-2. Time-course studies demonstrated high-level and productive infection. Studies performed in human iPSC-derived macrophages and primary monocyte-derived macrophages paralleled results seen in THP-1 cells but with lower infection levels. Up to 2% of macrophages were infected, with infected cells appearing multinucleated and syncytial. Addition of ruxolitinib, an inhibitor of JAK1/2 signalling, increased infection up to 10-fold, indicating limitation of infection through innate immune mechanisms. Sera from primary infections (n=80) mediated rare infection events, with a minority of samples (n=3) promoting significant infection. Competition assays confirmed results seen in sera, with the addition of neutralising mAbs diminishing the infection seen with infection-mediating mAbs. Thus, the presence of antibodies with potential to mediate infection is not sufficient to predict myeloid cell infection, rather, the context in which the antibodies are produced is key. Conclusion(s): We hypothesise that a nascent antibody response during peak viral replication in primary infection presents a window of opportunity for myeloid cells to become infected, while establishment of a robust polyclonal response via vaccination or prior infection reduces the likelihood of this occurring. Infection via antibody-FcR interactions could contribute to pathogenesis in primary infection, systemic virus spread or persistent infection.

Current Status and Prospects of Vaccines based on DDS Technology

The development of DDS technology has contributed critically to the unprecedentedly rapid requirement for vaccines against COVID-19. LNP-based mRNA vaccines represent a subset of emerging DDS technology. Despite the groundbreaking nature of these vaccines, they are yet to be perfected and as such, new technologies are being developed to optimize these vaccines. This review will focus on exploring one of the modalities of recombinant protein vaccines and will introduce various findings on the enhancement of vaccine efficacy using antigen modification technologies, including VLPs and Fc-fusion proteins, and adjuvant improvements.Alternate :抄録COVID-19に対してかつてない速度でワクチンが普及した背景には、DDS技術の発展が必要不可欠であった。特に、mRNAワクチンにおける脂質ナノ粒子（LNP）の開発は、まさにDDS技術の結集といえよう。一方で、mRNAワクチンを含め、現状のさまざまなワクチンは多くの課題を有しており、より効果的かつ安全なワクチン開発に資する基盤技術の確立が世界的に待望されている。本稿では、ワクチンモダリティの1つである組換えタンパク質ワクチンに焦点を絞り、抗原改変技術からアジュバントの改良に至るまで、ワクチン開発基盤技術の最新知見について紹介する。

Neurologic sequelae of COVID-19 are determined by immunologic imprinting from previous coronaviruses.

Coronavirus disease 2019 (COVID-19), which is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), remains a global public health emergency. Although SARS-CoV-2 is primarily a respiratory pathogen, extra-respiratory organs, including the central nervous system (CNS), can also be affected. Neurologic symptoms have been observed not only during acute SARS-CoV-2 infection, but also at distance from respiratory disease, also known as long-COVID or neurological post-acute sequelae of COVID-19 (neuroPASC). The pathogenesis of neuroPASC is not well understood, but hypotheses include SARS-CoV-2-induced immune dysfunctions, hormonal dysregulations, and persistence of SARS-CoV-2 reservoirs. In this prospective cohort study, we used a high throughput systems serology approach to dissect the humoral response to SARS-CoV-2 (and other common Coronaviruses - 229E, HKU1, NL63, OC43) in the serum and cerebrospinal fluid (CSF) from 112 infected individuals who developed (n = 18) or did not develop (n = 94) neuroPASC. Unique SARS-CoV-2 humoral profiles were observed in the CSF of neuroPASC, compared to serum responses. All antibody isotypes (IgG, IgM, IgA) and subclasses (IgA1-2; IgG1-4) were detected in serum, whereas CSF was characterized by focused IgG1 (and absence of IgM). These data argue in favor of compartmentalized brain-specific responses against SARS-CoV-2 through selective transfer of antibodies from the serum to the CSF across the blood-brain-barrier, rather than intrathecal synthesis, where more diversity in antibody classes/subclasses would be expected. Compared to individuals who did not develop post-acute complications following infection, individuals with neuroPASC had similar demographic features (median age 65 vs 66.5 years, respectively, p = 0.55; females 33% vs 44%, p = 0.52), but exhibited attenuated systemic antibody responses against SARS-CoV-2, characterized by decreased capacity to activate antibody-dependent complement deposition (ADCD), NK cell activation (ADNKA) and to bind Fcγ receptors. However, surprisingly, neuroPASC individuals showed significantly expanded antibody responses to other common Coronaviruses, including 229E, HKU1, NL63, and OC43. This biased humoral activation across coronaviruses was particularly enriched in neuroPASC individuals with poor outcome, suggesting an original antigenic sin (or immunologic imprinting), where pre-existing immune responses against related viruses shape the response to current infection, as a key prognostic marker of neuroPASC disease. Overall, these findings point to a pathogenic role for compromised anti-SARS-CoV-2 responses in the CSF, likely resulting in incomplete virus clearance from the brain and persistent neuroinflammation, in the development of post-acute neurologic complications of SARS-CoV-2 infection.

Unraveling and targeting the innate immune response in Multisystem Inflammatory Syndrome in Children (MIS-C)

OBJECTIVES/GOALS: The innate immune responses to Multisystem Inflammatory Syndrome in Children (MIS-C) are not fully known. Using samples from MIS-C, we will assess the cellular responses and develop a novel Tri-Specific Killer Engager (TRiKE) that engages innate immune cells to improve those responses. METHODS/STUDY POPULATION: We collected blood samples from 60 pediatric patients from which we isolated plasma and peripheral blood mononuclear cells. We received blood samples from 13 MIS-C, 32 severe acute COVID, 5 COVID-19 asymptomatic, and 15 COVID-19 negative patients. Using plasma, we then performed ELISAs to determine IgG antibody levels against SARS-CoV-2 and plaque reduction neutralization tests to determine neutralizing antibody functions. We isolated DNA to look at Fc receptor genetics. We also utilized utilize flow cytometry assays determine the phagocytosis and killing abilities of the innate cells from these patients. This data will be correlated with clinical outcomes. Additionally, we have developed a novel SARS-CoV-2 TRiKE which directs natural killer (NK) cell killing specifically to of COVID-19 infected cells. RESULTS/ANTICIPATED RESULTS: MIS-C patients had higher IgG antibody titers against SARS-CoV-2 compared to children with symptomatic or asymptomatic COVID. MIS-C patients also neutralized SARS-CoV-2 more effectively than children with acute symptomatic or asymptomatic COVID-19. We found natural killer cells and monocytes are dysfunctional in MIS-C patients and do not kill SARS-CoV-2 infected cells as well. Specifically, NK cells do not kill COVID-19 infected cells as well. To combat this, we have successfully generated and are now testing a Tri-Specific Killer engager (TRiKE) which binds one ends to NK cells, one end to the Spike protein on COVID-19 infected cells and contains IL-15 to improve NK cell function. We anticipate that we can improve NK cell killing of COVID-19 infected cells with this TRiKE. DISCUSSION/SIGNIFICANCE: We found that MIS-C patients have antibodies that can neutralize SARS-CoV-2 but that that innate immune cells that engage antibodies are dysfunctional. We are have successfully developed and are targeting this response with a TRiKE to improve innate immune cell functional;this may serve as an adjunctive therapeutic if proven successful.

The Therapeutic Effects of EFNB2-Fc in a Cell Model of Kawasaki Disease.

The EphrinB2/EphB4 signaling pathway involves the regulation of vascular morphogenesis and angiogenesis. However, little is known about EphrinB2/EphB4 in the pathogenesis of Kawasaki disease (KD) and coronary artery aneurysm formation. Hence, this study aimed to explore the role of EphrinB2/EphB4 and the potential therapeutic effect of EphrinB2-Fc in the coronary arterial endothelial injury of KD. The levels of EphB4 were compared between KD patients and healthy children. Human coronary artery endothelial cells (HCAECs) were stimulated with sera from acute KD patients to establish the KD cell model. The overexpression of EphB4 or treatment with EphrinB2-Fc was found to intervene in the cell model. The cell migration, angiogenesis, and proliferation ability were assessed, and the expression of inflammation-related factors was measured. Our study showed that EphB4 showed low expression in both KD patients and the cell model of KD. The EphB4 protein levels in the CECs of CAA+ KD patients were much lower than those in healthy children. EphrinB2-Fc treatment of KD sera-activated HCAECs suppressed cell proliferation, reduced the expression of inflammation-related factors (such as IL-6 and P-selectin), and elevated cell angiogenesis ability. The results reveal that EphrinB2-Fc has a protective function in endothelial cells and has promising clinical applications for protecting vascular endothelium in patients with KD.

Covid-19 prevention and treatment by targeting Fc-fusion proteins: An experience to fight emerging diseases.

The coronavirus disease 2019 (Covid-19) pandemic has been considered a major threat to human health. Effective therapeutic approaches are urgently required. Spike protein and the Angiotensin-converting enzyme 2 (ACE2) receptors have critical roles in SARS-CoV-2 infection. As a result, these two proteins are considered potential targets for the development of a wide variety of biotherapeutics and vaccines for controlling Covid-19. The fusion proteins have desirable medicinal properties, including high serum half-life, stability, and solubility in the body. Moreover, other Fc-fusion proteins used to treat other diseases have no known side effects. These Fc-fusion proteins are valuable biopharmaceuticals and have been proposed as therapeutic candidates for the treatment and prevention of Covid-19 owing to their potential therapeutic benefits.

Immune complexes as culprits of immunopathology in severe COVID-19.

Infection with the pandemic human coronavirus SARS-CoV-2 elicits a respiratory tract disease, termed Coronavirus disease 2019 (COVID-19). While a variable degree of disease-associated symptoms may emerge, severe COVID-19 is commonly associated with respiratory complications such as acute respiratory distress syndrome (ARDS), the necessity for mechanical ventilation or even extracorporeal membrane oxygenation (ECMO). Amongst others, disease outcome depends on age and pre-existing conditions like cardiovascular diseases, metabolic disorders but also age and biological sex. Intriguingly, increasing experimental and clinical evidence suggests that an exacerbated inflammatory response and in particular IgG immune complexes (ICs), significantly contribute to severe and prolonged COVID-19 disease progression. Vast amounts of deposited, unresolved ICs in tissue are capable to initiate an exaggerated Fc gamma receptor (FcγR) mediated signalling cascade which eventually results in common IC-associated organ diseases such as vasculitis, glomerulonephritis and arthritis, comorbidities that have been frequently reported for COVID-19. Moreover and independent of deposited ICs, very recent work identified soluble ICs (sIC) to be also present in the circulation of a majority of severely ill patients, where their systemic abundance correlated with disease severity. Thus, detection of circulating sICs in patients represents a potential marker for critical COVID-19 disease progression. Their detection early after clinical deterioration might become an indicator for the requirement of prompt anti-inflammatory treatment. Here, we review the role of ICs in COVID-19 progression, their possible origins and potential intervention strategies.

Single-domain antibody delivery using an mRNA platform protects against lethal doses of botulinum neurotoxin A.

Single-domain antibodies (sdAbs, VHHs, or nanobodies) are a promising tool for the treatment of both infectious and somatic diseases. Their small size greatly simplifies any genetic engineering manipulations. Such antibodies have the ability to bind hard-to-reach antigenic epitopes through long parts of the variable chains, the third complementarity-determining regions (CDR3s). VHH fusion with the canonical immunoglobulin Fc fragment allows the Fc-fusion single-domain antibodies (VHH-Fc) to significantly increase their neutralizing activity and serum half-life. Previously we have developed and characterized VHH-Fc specific to botulinum neurotoxin A (BoNT/A), that showed a 1000-fold higher protective activity than monomeric form when challenged with five times the lethal dose (5 LD50) of BoNT/A. During the COVID-19 pandemic, mRNA vaccines based on lipid nanoparticles (LNP) as a delivery system have become an important translational technology that has significantly accelerated the clinical introduction of mRNA platforms. We have developed an mRNA platform that provides long-term expression after both intramuscular and intravenous application. The platform has been extensively characterized using firefly luciferase (Fluc) as a reporter. An intramuscular administration of LNP-mRNA encoding VHH-Fc antibody made it possible to achieve its rapid expression in mice and resulted in 100% protection when challenged with up to 100 LD50 of BoNT/A. The presented approach for the delivery of sdAbs using mRNA technology greatly simplifies drug development for antibody therapy and can be used for emergency prophylaxis.

Enhancement of immunogenicity and neutralizing responses against SARS-CoV-2 spike protein using the Fc fusion fragment.

AIMS: Vaccination has played an important role in protecting against death and the severity of COVID-19. The recombinant protein vaccine platform has a long track record of safety and efficacy. Here, we fused the SARS-CoV-2 spike S1 subunit to the Fc region of IgG and investigated immunogenicity, reactivity to human vaccinated sera, and neutralizing activity as a candidate protein vaccine. MATERIALS AND METHOD: We evaluated the immunogenicity of CHO-expressed S1-Fc fusion protein and tag-free S1 protein in rabbits via the production of S1-specific polyclonal antibodies. We subsequently compared the neutralizing activities of sera from immunized rabbits and human-vaccinated individuals using a surrogate Virus Neutralization Test (sVNT). KEY FINDINGS: The results indicate that S1-specific polyclonal antibodies were induced in all groups; however, antibody levels were higher in rabbits immunized with S1-Fc fusion protein than tag-free S1 protein. Moreover, the reactivity of human vaccinated sera against S1-Fc fusion protein was significantly higher than tag-free S1 protein. Lastly, the results of the virus-neutralizing activity revealed that vaccination with S1-Fc fusion protein induced the highest level of neutralizing antibody response against SARS-CoV-2. SIGNIFICANCE: Our results demonstrate that the S1 protein accompanied by the Fc fragment significantly enhances the immunogenicity and neutralizing responses against SARS-CoV-2. It is hoped that this platform can be used for human vaccination.

Evaluation of the Mucosal Immunity Effect of Bovine Viral Diarrhea Virus Subunit Vaccine E2Fc and E2Ft.

Classified as a class B infectious disease by the World Organization for Animal Health (OIE), bovine viral diarrhea/mucosal disease is an acute, highly contagious disease caused by the bovine viral diarrhea virus (BVDV). Sporadic endemics of BVDV often lead to huge economic losses to the dairy and beef industries. To shed light on the prevention and control of BVDV, we developed two novel subunit vaccines by expressing bovine viral diarrhea virus E2 fusion recombinant proteins (E2Fc and E2Ft) through suspended HEK293 cells. We also evaluated the immune effects of the vaccines. The results showed that both subunit vaccines induced an intense mucosal immune response in calves. Mechanistically, E2Fc bonded to the Fc γ receptor (FcγRI) on antigen-presenting cells (APCs) and promoted IgA secretion, leading to a stronger T-cell immune response (Th1 type). The neutralizing antibody titer stimulated by the mucosal-immunized E2Fc subunit vaccine reached 1:64, which was higher than that of the E2Ft subunit vaccine and that of the intramuscular inactivated vaccine. The two novel subunit vaccines for mucosal immunity developed in this study, E2Fc and E2Ft, can be further used as new strategies to control BVDV by enhancing cellular and humoral immunity.

Intranasal Boosting with Spike Fc-RBD of Wild-Type SARS-CoV-2 Induces Neutralizing Antibodies against Omicron Subvariants and Reduces Viral Load in the Nasal Turbinate of Mice.

The emergence of new immune-evasive severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants and subvariants outpaces the development of vaccines specific against the dominant circulating strains. In terms of the only accepted immune correlate of protection, the inactivated whole-virion vaccine using wild-type SARS-CoV-2 spike induces a much lower serum neutralizing antibody titre against the Omicron subvariants. Since the inactivated vaccine given intramuscularly is one of the most commonly used coronavirus disease 2019 (COVID-19) vaccines in developing regions, we tested the hypothesis that intranasal boosting after intramuscular priming would provide a broader level of protection. Here, we showed that one or two intranasal boosts with the Fc-linked trimeric spike receptor-binding domain from wild-type SARS-CoV-2 can induce significantly higher serum neutralizing antibodies against wild-type SARS-CoV-2 and the Omicron subvariants, including BA.5.2 and XBB.1, with a lower titre in the bronchoalveolar lavage of vaccinated Balb/c mice than vaccination with four intramuscular doses of inactivated whole virion vaccine. The intranasally vaccinated K18-hACE2-transgenic mice also had a significantly lower nasal turbinate viral load, suggesting a better protection of the upper airway, which is the predilected site of infection by Omicron subvariants. This intramuscular priming and intranasal boosting approach that achieves broader cross-protection against Omicron variants and subvariants may lengthen the interval required for changing the vaccine immunogen from months to years.

A randomized phase I/II safety and immunogenicity study of the Montanide-adjuvanted SARS-CoV-2 spike protein-RBD-Fc vaccine, AKS-452.

BACKGROUND: Previous interim data from a phase I study of AKS-452, a subunit vaccine comprising an Fc fusion of the respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein receptor binding domain (SP/RBD) emulsified in the water-in-oil adjuvant, Montanide™ ISA 720, suggested a good safety and immunogenicity profile in healthy adults. This phase I study was completed and two dosing regimens were further evaluated in this phase II study. METHODS: This phase II randomized, open-labelled, parallel group study was conducted at a single site in The Netherlands with 52 healthy adults (18 - 72 years) receiving AKS-452 subcutaneously at one 90 µg dose (cohort 1, 26 subjects) or two 45 µg doses 28 days apart (cohort 2, 26 subjects). Serum samples were collected at the first dose (day 0) and at days 28, 56, 90, and 180. Safety and immunogenicity endpoints were assessed, along with induction of IgG isotypes, cross-reactive immunity against viral variants, and IFN-γ T cell responses. RESULTS: All AEs were mild/moderate (grades 1 or 2), and no SAEs were attributable to AKS-452. Seroconversion rates reached 100% in both cohorts, although cohort 2 showed greater geometric mean IgG titers that were stable through day 180 and associated with enhanced potencies of SP/RBD-ACE2 binding inhibition and live virus neutralization. AKS-452-induced IgG titers strongly bound mutant SP/RBD from several SARS-CoV-2 variants (including Omicrons) that were predominantly of the favorable IgG1/3 isotype and IFN-γ-producing T cell phenotype. CONCLUSION: These favorable safety and immunogenicity profiles of the candidate vaccine as demonstrated in this phase II study are consistent with those of the phase I study (ClinicalTrials.gov: NCT04681092) and suggest that a total of 90 µg received in 2 doses may offer a greater duration of cross-reactive neutralizing titers than when given in a single dose.

Immunogenic fusion proteins induce neutralizing SARS-CoV-2 antibodies in the serum and milk of sheep.

Antigen-specific polyclonal immunoglobulins derived from the serum, colostrum, or milk of immunized ruminant animals have potential as scalable therapeutics for the control of viral diseases including COVID-19. Here we show that the immunization of sheep with fusions of the SARS-CoV-2 receptor binding domain (RBD) to ovine IgG2a Fc domains promotes significantly higher levels of antigen-specific antibodies compared to native RBD or full-length spike antigens. This antibody population contained elevated levels of neutralizing antibodies that suppressed binding between the RBD and hACE2 receptors in vitro. A second immune-stimulating fusion candidate, Granulocyte-macrophage colony-stimulating factor (GM-CSF), induced high neutralizing responses in select animals but narrowly missed achieving significance. We further demonstrated that the antibodies induced by these fusion antigens were transferred into colostrum/milk and possessed cross-neutralizing activity against diverse SARS-CoV-2 variants. Our findings highlight a new pathway for recombinant antigen design in ruminant animals with applications in immune milk production and animal health.

Subclass-switched anti-spike IgG3 oligoclonal cocktails strongly enhance Fc-mediated opsonization.

Antibodies play a central role in the immune defense against SARS-CoV-2. Emerging evidence has shown that nonneutralizing antibodies are important for immune defense through Fc-mediated effector functions. Antibody subclass is known to affect downstream Fc function. However, whether the antibody subclass plays a role in anti-SARS-CoV-2 immunity remains unclear. Here, we subclass-switched eight human IgG1 anti-spike monoclonal antibodies (mAbs) to the IgG3 subclass by exchanging their constant domains. The IgG3 mAbs exhibited altered avidities to the spike protein and more potent Fc-mediated phagocytosis and complement activation than their IgG1 counterparts. Moreover, combining mAbs into oligoclonal cocktails led to enhanced Fc- and complement receptor-mediated phagocytosis, superior to even the most potent single IgG3 mAb when compared at equivalent concentrations. Finally, in an in vivo model, we show that opsonic mAbs of both subclasses can be protective against a SARS-CoV-2 infection, despite the antibodies being nonneutralizing. Our results suggest that opsonic IgG3 oligoclonal cocktails are a promising idea to explore for therapy against SARS-CoV-2, its emerging variants, and potentially other viruses.