Internalization of HIV-1 by Phagocytes Is Increased When Virions Are Opsonized with Multimeric Antibody in the Presence of Complement.

The low abundance of envelope spikes and the inability of IgG to aggregate virions render HIV-1 an inadequate target for antibody-mediated clearance by phagocytes. In an attempt to improve the ability of antibody to mediate the internalization of HIV-1 virions, we generated multimers of the broadly neutralizing HIV-1-specific monoclonal antibody (MAb) VRC01 using site-directed mutagenesis of the Fc segment. We then measured virion internalization using primary human monocytes and neutrophils. We found that, in the absence of complement, immune complexes consisting of HIV-1 virions and VRC01 multimers were slightly more efficiently internalized than were complexes formed with monomeric VRC01. The presence of complement, however, greatly augmented internalization of immune complexes formed with the multimeric MAb but had little impact on monomeric MAb-mediated internalization. Multimerization and the presence of complement overcome the limited ability of monomeric antibody to mediate internalization of HIV-1 virions and may thus provide a therapeutic approach to clearing virus. IMPORTANCE Antibody-mediated internalization of HIV-1 by phagocytes, a potential mechanism for clearing virus, is very inefficient. In an effort to improve viral clearance, we produced a multimeric form of the broadly neutralizing monoclonal antibody VRC01. We found that VRC01 antibody multimers (primarily hexamers) were only slightly more efficient in mediating HIV-1 internalization than was monomeric VRC01. However, the addition of complement resulted in substantially greater internalization of multimer-opsonized virus. In contrast, complement had little if any impact on internalization of monomer-opsonized virus. Therefore, antibody multimerization in combination with complement may overcome the limited ability of monomeric antibody to mediate internalization of HIV-1 virions. Our findings may provide a therapeutic approach to clearing virus.

Vaccination against SARS-CoV-2 using extracellular blebs derived from spike protein-expressing dendritic cells.

COVID-19 has caused significant morbidity and mortality worldwide but also accelerated the clinical use of emerging vaccine formulations. To address the current shortcomings in the prevention and treatment of SARS-CoV-2 infection, this study developed a novel vaccine platform that closely mimics dendritic cells (DCs) in antigen presentation and T-cell stimulation in a cell-free and tunable manner. Genetically engineered DCs that express the SARS-CoV-2 spike protein (S) were chemically converted into extracellular blebs (EBs). The resulting EBs elicited potentially protective humoral immunity in vivo, indicated by the production of antibodies that potently neutralized S-pseudotyped virus, presenting EBs as a promising and safe vaccine.

Virus Control in Vaccinated Rhesus Macaques Is Associated with Neutralizing and Capturing Antibodies against the SHIV Challenge Virus but Not with V1V2 Vaccine-Induced Anti-V2 Antibodies Alone.

The role of vaccine-induced anti-V2 Abs was tested in three protection experiments in rhesus macaques. In an experiment using immunogens similar to those in the RV144 vaccine trial (Anti-envelope [Env]), nine rhesus macaques were coimmunized with gp16092TH023 DNA and SIV gag and gp120A244 and gp120MN proteins. In two V2-focused experiments (Anti-V2 and Anti-V2 Mucosal), nine macaques in each group were immunized with V1V292TH023 DNA, V1V2A244 and V1V2CasaeA2 proteins, and cyclic V2CaseA2 peptide. DNA and protein immunogens, formulated in Adjuplex, were given at 0, 4, 12, and 20 weeks, followed by intrarectal SHIVBaL.P4 challenges. Peak plasma viral loads (PVL) of 106-107 copies/ml developed in all nine sham controls. Overall, PVL was undetectable in one third of immunized macaques, and two animals tightly controlled the virus with the Anti-V2 Mucosal vaccine strategy. In the Anti-Env study, Abs that captured or neutralized SHIVBaL.P4 inversely correlated with PVL. Conversely, no correlation with PVL was found in the Anti-V2 experiments with nonneutralizing plasma Abs that only captured virus weakly. Titers of Abs against eight V1V2 scaffolds and cyclic V2 peptides were comparable between controllers and noncontrollers as were Ab-dependent cellular cytotoxicity and Ab-dependent cell-mediated virus inhibition activities against SHIV-infected target cells and phagocytosis of gp120-coated beads. The Anti-Env experiment supports the role of vaccine-elicited neutralizing and nonneutralizing Abs in control of PVL. However, the two V2-focused experiments did not support a role for nonneutralizing V2 Abs alone in controlling PVL, as neither Ab-dependent cellular cytotoxicity, Ab-dependent cell-mediated virus inhibition, nor phagocytosis correlated inversely with heterologous SHIVBaL.P4 infection.

Diverse antiviral IgG effector activities are predicted by unique biophysical antibody features.

BACKGROUND: The critical role of antibody Fc-mediated effector functions in immune defense has been widely reported in various viral infections. These effector functions confer cellular responses through engagement with innate immune cells. The precise mechanism(s) by which immunoglobulin G (IgG) Fc domain and cognate receptors may afford protection are poorly understood, however, in the context of HIV/SHIV infections. Many different in vitro assays have been developed and utilized to measure effector functions, but the extent to which these assays capture distinct antibody activities has not been fully elucidated. RESULTS: In this study, six Fc-mediated effector function assays and two biophysical antibody profiling assays were performed on a common set of samples from HIV-1 infected and vaccinated subjects. Biophysical antibody profiles supported robust prediction of diverse IgG effector functions across distinct Fc-mediated effector function assays. While a number of assays showed correlated activities, supervised machine learning models indicated unique antibody features as primary contributing factors to the associated effector functions. Additional experiments established the mechanistic relevance of relationships discovered using this unbiased approach. CONCLUSIONS: In sum, this study provides better resolution on the diversity and complexity of effector function assays, offering a clearer perspective into this family of antibody mechanisms of action to inform future HIV-1 treatment and vaccination strategies.

Impact of Th1 CD4 Follicular Helper T Cell Skewing on Antibody Responses to an HIV-1 Vaccine in Rhesus Macaques.

Generating durable humoral immunity through vaccination depends upon effective interactions of follicular helper T (Tfh) cells with germinal center (GC) B cells. Th1 polarization of Tfh cells is an important process shaping the success of Tfh-GC B cell interactions by influencing costimulatory and cytokine-dependent Tfh help to B cells. However, the question remains as to whether adjuvant-dependent modulation of Tfh cells enhances HIV-1 vaccine-induced antienvelope (anti-Env) antibody responses. We investigated whether an HIV-1 vaccine platform designed to increase the number of Th1-polarized Tfh cells enhances the magnitude and quality of anti-Env antibodies. Utilizing a novel interferon-induced protein 10 (IP-10)-adjuvanted HIV-1 DNA prime followed by a monophosphoryl lipid A and QS-21 (MPLA+QS-21)-adjuvanted Env protein boost (DIP-10 PALFQ) in macaques, we observed higher anti-Env serum IgG titers with greater cross-clade reactivity, specificity for V1V2, and effector functions than in macaques primed with DNA lacking IP-10 and boosted with MPLA-plus-alum-adjuvanted Env protein (DPALFA) The DIP-10 PALFQ vaccine regimen elicited higher anti-Env IgG1 and lower IgG4 antibody levels in serum, showing for the first time that adjuvants can dramatically impact the IgG subclass profile in macaques. The DIP-10 PALFQ regimen also increased vaginal and rectal IgA antibodies to a greater extent. Within lymph nodes, we observed augmented GC B cell responses and the promotion of Th1 gene expression profiles in GC Tfh cells. The frequency of GC Tfh cells correlated with both the magnitude and avidity of anti-Env serum IgG. Together, these data suggest that adjuvant-induced stimulation of Th1-Tfh cells is an effective strategy for enhancing the magnitude and quality of anti-Env antibody responses.IMPORTANCE The results of the RV144 trial demonstrated that vaccination could prevent HIV transmission in humans and that longevity of anti-Env antibodies may be key to this protection. Efforts to improve upon the prime-boost vaccine regimen used in RV144 have indicated that booster immunizations can increase serum anti-Env antibody titers but only transiently. Poor antibody durability hampers efforts to develop an effective HIV-1 vaccine. This study was designed to identify the specific elements involved in the immunological mechanism necessary to produce robust HIV-1-specific antibodies in rhesus macaques. By clearly defining immune-mediated pathways that improve the magnitude and functionality of the anti-HIV-1 antibody response, we will have the foundation necessary for the rational development of an HIV-1 vaccine.

Antibody Responses Elicited by Immunization with BG505 Trimer Immune Complexes.

Immune complex (IC) vaccines have been successfully used to increase immune responses against various pathogens, including HIV-1. Additionally, IC vaccines can induce qualitatively different antibody responses, with distinct antigenic specificities compared to the same antigens used alone. Here we measured the HIV-1-specific antibody responses in female New Zealand White rabbits after immunization with ICs made from BG505 SOSIP.664 trimers (BG505 trimers) and three rabbit monoclonal antibodies (MAbs) with different neutralization profiles. Two of the MAbs were specific for a hole in the glycan shield of the BG505 trimer, while the third, which bound less avidly, was specific for determinants at the gp41-gp120 interface. We found that immunization with one of the glycan-hole-specific ICs resulted in lower levels of trimer-binding antibodies compared to vaccination with the uncomplexed trimer, and that ICs made using either of the glycan-hole-specific MAbs resulted in lower rates of anti-trimer antibody decay. We concluded that ICs based on MAbs that bound to the immunodominant glycan hole epitope likely diverted antibody responses, to some extent, away from this site and to other regions of the trimer. However, this outcome was not accompanied by a widening of the breadth or an increase in the potency of neutralizing antibody responses compared with uncomplexed trimers.IMPORTANCE Immunodominant epitopes may suppress immune responses to more desirable determinants, such as those that elicit potentially protective neutralizing antibody responses. To overcome this problem, we attempted to mask immunodominant glycan holes by immunizing rabbits with ICs consisting of the BG505 SOSIP.664 gp140 trimer and MAbs that targeted the glycan holes. We found that IC vaccination likely diverted antibody responses, to some extent, away from the glycan holes and toward other regions of the trimer. IC vaccination resulted in slower decay of HIV-1-specific antibodies than did immunization with uncomplexed trimer. We did not observe a widening of the breadth or an increase in the potency of neutralizing antibody responses compared to uncomplexed trimers. Our results suggest that selective epitope dampening of BG505 trimers by ICs is rather ineffective. However, IC vaccination may represent a novel means of increasing the duration of vaccine-induced antibody responses.

Combination Adenovirus and Protein Vaccines Prevent Infection or Reduce Viral Burden after Heterologous Clade C Simian-Human Immunodeficiency Virus Mucosal Challenge.

HIV vaccine development is focused on designing immunogens and delivery methods that elicit protective immunity. We evaluated a combination of adenovirus (Ad) vectors expressing HIV 1086.C (clade C) envelope glycoprotein (Env), SIV Gag p55, and human pegivirus GBV-C E2 glycoprotein. We compared replicating simian (SAd7) with nonreplicating human (Ad4) adenovirus-vectored vaccines paired with recombinant proteins in a novel prime-boost regimen in rhesus macaques, with the goal of eliciting protective immunity against SHIV challenge. In both vaccine groups, plasma and buccal Env-specific IgG, tier 1 heterologous neutralizing antibodies, and antibody-dependent cell-mediated viral inhibition were readily generated. High Env-specific T cell responses elicited in all vaccinees were significantly greater than responses targeting Gag. After three intrarectal exposures to heterologous tier 1 clade C SHIV, all 10 sham-vaccinated controls were infected, whereas 4/10 SAd7- and 3/10 Ad4-vaccinated macaques remained uninfected or maintained tightly controlled plasma viremia. Time to infection was significantly delayed in SAd7-vaccinated macaques compared to the controls. Cell-associated and plasma virus levels were significantly lower in each group of vaccinated macaques compared to controls; the lowest plasma viral burden was found in animals vaccinated with the SAd7 vectors, suggesting superior immunity conferred by the replicating simian vectors. Furthermore, higher V1V2-specific binding antibody titers correlated with viral control in the SAd7 vaccine group. Thus, recombinant Ad plus protein vaccines generated humoral and cellular immunity that was effective in either protecting from SHIV acquisition or significantly reducing viremia in animals that became infected, consequently supporting additional development of replicating Ad vectors as HIV vaccines.IMPORTANCE There is a well-acknowledged need for an effective AIDS vaccine that protects against HIV infection and limits in vivo viral replication and associated pathogenesis. Although replicating virus vectors have been advanced as HIV vaccine platforms, there have not been any direct comparisons of the replicating to the nonreplicating format. The present study directly compared the replicating SAd7 to nonreplicating Ad4 vectors in macaques and demonstrated that in the SAd7 vaccine group, the time to infection was significantly delayed compared to the control group, and V1V2 Env-specific binding antibodies correlated with viral outcomes. Viral control was significantly enhanced in vaccinated macaques compared to controls, and in infected SAd7-vaccinated macaques compared to Ad4-vaccinated macaques, suggesting that this vector may have conferred more effective immunity. Because blocking infection is so difficult with current vaccines, development of a vaccine that can limit viremia if infection occurs would be valuable. These data support further development of replicating adenovirus vectors.

Human immunodeficiency virus type-1 (HIV-1) evades antibody-dependent phagocytosis.

Fc gamma receptor (FcyR)-mediated antibody functions play a crucial role in preventing HIV infection. One such function, antibody-dependent phagocytosis (ADP), is thought to be involved in controlling other viral infections, but its role in HIV infection is unknown. We measured the ability of HIV-specific polyclonal and monoclonal antibodies (mAbs) to mediate the internalization of HIV-1 virions and HIV-1-decorated cells by phagocytes. To measure ADP of virions, we primarily used a green-fluorescent protein-expressing molecular clone of HIV-1JRFL, an R5, clinical isolate, in combination with polyclonal HIVIG or mAbs known to capture and/or neutralize HIV-1. THP-1 and U937 cells, as well as freshly isolated primary monocytes from healthy individuals, were used as phagocytic effector cells, and uptake of virions was measured by cytometry. We surprisingly found minimal or no ADP of virions with any of the antibodies. However, after coating virions with gp41 or with gp41-derived peptides, gp41- (but not gp120-) specific mAbs efficiently mediated phagocytosis. We estimated that a minimum of a few hundred gp41 molecules were needed for successful phagocytosis, which is similar to the number of envelope spikes on viruses that are readily phagocytosed (e.g. influenza virus). Furthermore, by employing fluorescence correlation spectroscopy, a well-established technique to measure particle sizes and aggregation phenomena, we found a clear association between virus aggregation and ADP. In contrast to virions themselves, virion-decorated cells were targets for ADP or trogocytosis in the presence of HIV-specific antibodies. Our findings indicate that ADP of virions may not play a role in preventing HIV infection, likely due to the paucity of trimers and the consequent inability of virion-bound antibody to cross-link FcyRs on phagocytes. However, ADP or trogocytosis could play a role in clearing HIV-infected cells and cells on the verge of infection.

Glycan modulation and sulfoengineering of anti-HIV-1 monoclonal antibody PG9 in plants.

Broadly neutralizing anti-HIV-1 monoclonal antibodies, such as PG9, and its derivative RSH hold great promise in AIDS therapy and prevention. An important feature related to the exceptional efficacy of PG9 and RSH is the presence of sulfated tyrosine residues in their antigen-binding regions. To maximize antibody functionalities, we have now produced glycan-optimized, fucose-free versions of PG9 and RSH in Nicotiana benthamiana. Both antibodies were efficiently sulfated in planta on coexpression of an engineered human tyrosylprotein sulfotransferase, resulting in antigen-binding and virus neutralization activities equivalent to PG9 synthesized by mammalian cells ((CHO)PG9). Based on the controlled production of both sulfated and nonsulfated variants in plants, we could unequivocally prove that tyrosine sulfation is critical for the potency of PG9 and RSH. Moreover, the fucose-free antibodies generated in N. benthamiana are capable of inducing antibody-dependent cellular cytotoxicity, an activity not observed for (CHO)PG9. Thus, tailoring of the antigen-binding site combined with glycan modulation and sulfoengineering yielded plant-produced anti-HIV-1 antibodies with effector functions superior to PG9 made in CHO cells.

Relationship between Vaccine-Induced Antibody Capture of Infectious Virus and Infection Outcomes following Repeated Low-Dose Rectal Challenges with Simian Immunodeficiency Virus SIVmac251.

UNLABELLED: Antibodies are known to enhance in vitro infection by human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV). We measured the ability of antibodies induced by ALVAC-SIV/gp120 vaccination, given with alum or MF59 adjuvant, to capture infectious SIVmac251 and determined the association between capture and infection outcomes following low-dose, repeated rectal challenge of rhesus macaques. We found that capture correlated with the number of transmitted/founder (T/F) variants that established infection, such that animals whose plasma captured more virus were infected with a higher number of T/F strains. Capture also correlated with results of Env binding assays, indicating that greater immunogenicity resulted in greater capture. Although vaccination elicited negligible neutralizing activity against the challenge strain (50% inhibitory dilutions of >1/80 in all cases), animals with low capture and whose plasma, at a fixed dilution, inhibited a higher fraction of virus were infected at a lower rate than animals with high capture and low neutralization (P = 0.039); only animals with the low capture/high neutralization response profile were protected compared with unvaccinated control animals (P = 0.026). In a sieve analysis, high capture and low capture were distinguishable on the basis of polymorphisms in the V1 loop of Env at amino acids 144 and 145. Our results indicate that vaccine-induced antibody that binds to and captures infectious virus but does not inhibit its infectivity may enhance the likelihood of infection following rectal challenge with SIVmac251. Higher immunogenicity resulting in better antibody capture but similar anti-infectivity may not improve vaccine efficacy. IMPORTANCE: Vaccines generally prevent viral infections by eliciting antibodies that inhibit virus infectivity. However, antibodies, including those induced by vaccination, have the potential to enhance, rather than prevent infection. We measured the ability of vaccine-induced antibodies to capture infectious simian immunodeficiency virus (SIV) and explored the relationship between virus capture and infection outcomes. We found that capture correlated with the number of SIV variants that established infection, such that animals whose plasma captured more virus were infected with a higher number of unique strains. In addition, animals whose sera had high capture but weak anti-infectivity activity were infected at a higher rate than were animals with low capture and stronger anti-infectivity activity. These results suggest that vaccines that induce antibodies that bind to and capture infectious virus but do not inhibit virus infectivity will not be effective in preventing infection.

Introduction of germline residues improves the stability of anti-HIV mAb 2G12-IgM.

Immunoglobulins M (IgMs) are gaining increasing attention as biopharmaceuticals since their multivalent mode of binding can give rise to high avidity. Furthermore, IgMs are potent activators of the complement system. However, they are frequently difficult to express recombinantly and can suffer from low conformational stability. Here, the broadly neutralizing anti-HIV-1 antibody 2G12 was class-switched to IgM and then further engineered by introduction of 17 germline residues. The impact of these changes on the structure and conformational stability of the antibody was then assessed using a range of biophysical techniques. We also investigated the effects of the class switch and germline substitutions on the ligand-binding properties of 2G12 and its capacity for HIV-1 neutralization. Our results demonstrate that the introduced germline residues improve the conformational and thermal stability of 2G12-IgM without altering its overall shape and ligand-binding properties. Interestingly, the engineered protein was found to exhibit much lower neutralization potency than its wild-type counterpart, indicating that potent antigen recognition is not solely responsible for IgM-mediated HIV-1 inactivation.

Enhanced in vitro transcytosis of simian immunodeficiency virus mediated by vaccine-induced antibody predicts transmitted/founder strain number after rectal challenge.

BACKGROUND: The time to acquisition of simian immunodeficiency virus (SIV) infection following low-dose repeated rectal challenge correlated inversely with the number of transmitted/founder strains among macaques vaccinated with ALVAC-SIV/gp120 or gp120 alone. We determined if the ability of postvaccination, prechallenge sera to enhance SIVmac251 transcytosis across epithelial cells was associated with transmitted/founder strain number. METHODS: Transcytosis was carried out by exposing sera and SIVmac251 to the apical surface of human endometrial carcinoma (HEC-1A) cells at pH 6.0 and 12 hours later quantifying virus in fluid bathing the basolateral cell surface (maintained at pH 7.4). These conditions allow Fc neonatal receptor (FcRn)-dependent shuttling of virus across cells. RESULTS: There was a strong correlation between the amount of virus transcytosed and number of transmitted variants (R = 0.86, P < .0001). We also found that 4 animals who remained uninfected after repeated rectal challenges had lower serum transcytosis activity than did 19 animals who subsequently became infected (P = .003). Using immunohistochemistry, we demonstrated FcRn on columnar epithelial cells facing the lumen of the macaque rectum. CONCLUSIONS: Vaccine-induced antibody capable of enhancing transcytosis in vitro via FcRn may play a role in determining transmitted/founder strain number and infection outcomes following in vivo challenge.

The Neonatal Fc receptor (FcRn) enhances human immunodeficiency virus type 1 (HIV-1) transcytosis across epithelial cells.

The mechanisms by which human immunodeficiency virus type 1 (HIV-1) crosses mucosal surfaces to establish infection are unknown. Acidic genital secretions of HIV-1-infected women contain HIV-1 likely coated by antibody. We found that the combination of acidic pH and Env-specific IgG, including that from cervicovaginal and seminal fluids of HIV-1-infected individuals, augmented transcytosis across epithelial cells as much as 20-fold compared with Env-specific IgG at neutral pH or non-specific IgG at either pH. Enhanced transcytosis was observed with clinical HIV-1 isolates, including transmitted/founder strains, and was eliminated in Fc neonatal receptor (FcRn)-knockdown epithelial cells. Non-neutralizing antibodies allowed similar or less transcytosis than neutralizing antibodies. However, the ratio of total:infectious virus was higher for neutralizing antibodies, indicating that they allowed transcytosis while blocking infectivity of transcytosed virus. Immunocytochemistry revealed abundant FcRn expression in columnar epithelia lining the human endocervix and penile urethra. Acidity and Env-specific IgG enhance transcytosis of virus across epithelial cells via FcRn and could facilitate translocation of virus to susceptible target cells following sexual exposure.

Neutralizing the threat: harnessing broadly neutralizing antibodies against HIV-1 for treatment and prevention.

Broadly neutralizing antibodies (bnAbs) targeting the human immunodeficiency virus-1 (HIV-1) have played a crucial role in elucidating and characterizing neutralization-sensitive sites on the HIV-1 envelope spike and in informing vaccine development. Continual advancements in identifying more potent bnAbs, along with their capacity to trigger antibody-mediated effector functions, coupled with modifications to extend their half-life, position them as promising candidates for both HIV-1 treatment and prevention. While current pharmacological interventions have made significant progress in managing HIV-1 infection and enhancing quality of life, no definitive cure or vaccines have been developed thus far. Standard treatments involve daily oral anti-retroviral therapy, which, despite its efficacy, can lead to notable long-term side effects. Recent clinical trial data have demonstrated encouraging therapeutic and preventive potential for bnAb therapies in both HIV-1-infected individuals and those without the infection. This review provides an overview of the advancements in HIV-1-specific bnAbs and discusses the insights gathered from recent clinical trials regarding their application in treating and preventing HIV-1 infection.

A genome-edited N. benthamiana line for industrial-scale production of recombinant glycoproteins with targeted N-glycosylation.

Control over glycosylation is an important quality parameter in recombinant protein production. Here, we demonstrate the generation of a marker-free genome edited Nicotiana benthamiana N-glycosylation mutant (NbXF-KO) carrying inactivated ß1,2-xylosyltransferase and α1,3-fucosyltransferase genes. The knockout of seven genes and their stable inheritance was confirmed by DNA sequencing. Mass spectrometric analyses showed the synthesis of N-glycans devoid of plant-specific ß1,2-xylose and core α 1,3-fucose on endogenous proteins and a series of recombinantly expressed glycoproteins with different complexities. Further transient glycan engineering towards more diverse human-type N-glycans resulted in the production of recombinant proteins decorated with ß1,4-galactosylated and α2,6-sialylated structures, respectively. Notably, a monoclonal antibody expressed in the NbXF-KO displayed glycosylation-dependent activities. Collectively, the engineered plants grow normally and are well suited for upscaling, thereby meeting industrial and regulatory requirements for the production of high-quality therapeutic proteins.

SARS-CoV-2 infection of endothelial cells, dependent on flow-induced ACE2 expression, drives hypercytokinemia in a vascularized microphysiological system.

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), responsible for COVID-19, has caused nearly 7 million deaths worldwide. Severe cases are marked by an aggressive inflammatory response known as hypercytokinemia, contributing to endothelial damage. Although vaccination has reduced hospitalizations, hypercytokinemia persists in breakthrough infections, emphasizing the need for disease models mimicking this response. Using a 3D microphysiological system (MPS), we explored the vascular role in SARS-CoV-2-induced hypercytokinemia. Methods: The vascularized micro-organ (VMO) MPS, consisting of human-derived primary endothelial cells (ECs) and stromal cells within an extracellular matrix, was used to model SARS-CoV-2 infection. A non-replicative pseudotyped virus fused to GFP was employed, allowing visualization of viral entry into human ECs under physiologic flow conditions. Expression of ACE2, TMPRSS2, and AGTR1 was analyzed, and the impact of viral infection on ACE2 expression, vascular inflammation, and vascular morphology was assessed. Results: The VMO platform facilitated the study of COVID-19 vasculature infection, revealing that ACE2 expression increased significantly in direct response to shear stress, thereby enhancing susceptibility to infection by pseudotyped SARS-CoV-2. Infected ECs secreted pro-inflammatory cytokines, including IL-6 along with coagulation factors. Cytokines released by infected cells were able to activate downstream, non-infected EC, providing an amplification mechanism for inflammation and coagulopathy. Discussion: Our findings highlight the crucial role of vasculature in COVID-19 pathogenesis, emphasizing the significance of flow-induced ACE2 expression and subsequent inflammatory responses. The VMO provides a valuable tool for studying SARS-CoV-2 infection dynamics and evaluating potential therapeutics.

An oligosaccharyltransferase from Leishmania donovani increases the N-glycan occupancy on plant-produced IgG1.

N-Glycosylation of immunoglobulin G1 (IgG1) at the heavy chain Fc domain (Asn297) plays an important role for antibody structure and effector functions. While numerous recombinant IgG1 antibodies have been successfully expressed in plants, they frequently display a considerable amount (up to 50%) of unglycosylated Fc domain. To overcome this limitation, we tested a single-subunit oligosaccharyltransferase from the protozoan Leishmania donovani (LdOST) for its ability to improve IgG1 Fc glycosylation. LdOST fused to a fluorescent protein was transiently expressed in Nicotiana benthamiana and confocal microscopy confirmed the subcellular location at the endoplasmic reticulum. Transient co-expression of LdOST with two different IgG1 antibodies resulted in a significant increase (up to 97%) of Fc glycosylation while leaving the overall N-glycan composition unmodified, as determined by different mass spectrometry approaches. While biochemical and functional features of "glycosylation improved" antibodies remained unchanged, a slight increase in FcγRIIIa binding and thermal stability was observed. Collectively, our results reveal that LdOST expression is suitable to reduce the heterogeneity of plant-produced antibodies and can contribute to improving their stability and effector functions.

A limited number of antibody specificities mediate broad and potent serum neutralization in selected HIV-1 infected individuals.

A protective vaccine against HIV-1 will likely require the elicitation of a broadly neutralizing antibody (bNAb) response. Although the development of an immunogen that elicits such antibodies remains elusive, a proportion of HIV-1 infected individuals evolve broadly neutralizing serum responses over time, demonstrating that the human immune system can recognize and generate NAbs to conserved epitopes on the virus. Understanding the specificities that mediate broad neutralization will provide insight into which epitopes should be targeted for immunogen design and aid in the isolation of broadly neutralizing monoclonal antibodies from these donors. Here, we have used a number of new and established technologies to map the bNAb specificities in the sera of 19 donors who exhibit among the most potent cross-clade serum neutralizing activities observed to date. The results suggest that broad and potent serum neutralization arises in most donors through a limited number of specificities (1-2 per donor). The major targets recognized are an epitope defined by the bNAbs PG9 and PG16 that is associated with conserved regions of the V1, V2 and V3 loops, an epitope overlapping the CD4 binding site and possibly the coreceptor binding site, an epitope sensitive to a loss of the glycan at N332 and distinct from that recognized by the bNAb 2G12 and an epitope sensitive to an I165A substitution. In approximately half of the donors, key N-linked glycans were critical for expression of the epitopes recognized by the bNAb specificities in the sera.

Fc-glycosylation influences Fcγ receptor binding and cell-mediated anti-HIV activity of monoclonal antibody 2G12.

Interactions between the Fc segment of IgG and FcγRs on a variety of cells are likely to play an important role in the anti-HIV activity of Abs. Because the nature of the glycan structure on the Fc domain is a critical determinant of Fc-FcγR binding, proper Fc glycosylation may contribute to Ab-mediated protection. We have generated five different glycoforms of the broadly HIV-1-neutralizing mAb 2G12 in wild-type and glycoengineered plants and Chinese hamster ovary cells. Plant-derived 2G12 exhibited highly homogeneous glycosylation profiles with a single dominant N-glycan species. Using flow cytometry with FcγR-expressing cell lines, all 2G12 glycoforms demonstrated similar binding to FcγRI, FcγRIIa, and FcγRIIb. In contrast, two glycoforms derived from glycoengineered plants that lack plant-specific xylose and core α1,3-fucose, and instead carry human-like glycosylation with great uniformity, showed significantly enhanced binding to FcγRIIIa compared with Chinese hamster ovary or wild-type plant-derived 2G12. Using surface plasmon resonance, we show that binding of 2G12 to FcγRIIIa is markedly affected by core fucose, irrespective of its plant-specific α1,3 or mammalian-type α1,6 linkage. Consistent with this finding, 2G12 glycoforms lacking core fucose (and xylose) mediated higher antiviral activity against HIV-1 or simian immunodeficiency virus as measured by Ab-dependent cell-mediated virus inhibition. This is, to our knowledge, the first demonstration that specific alterations of Fc glycosylation can improve antiviral activity. Such alterations may result in better immunotherapeutic reagents. Moreover, biasing vaccine-induced immune responses toward optimal Fc glycosylation patterns could result in improved vaccine efficacy.