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 (CHOPG9). 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 CHOPG9. 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.

Download Full-text

Highly active engineered IgG3 antibodies against SARS-CoV-2

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2107249118 ◽

2021 ◽

Vol 118 (42) ◽

pp. e2107249118

Author(s):

Somanath Kallolimath ◽

Lin Sun ◽

Roman Palt ◽

Karin Stiasny ◽

Patrick Mayrhofer ◽

...

Keyword(s):

Nicotiana Benthamiana ◽

Vaccine Development ◽

Binding Activity ◽

The Other ◽

Cross Linking ◽

Antigen Binding ◽

In Planta ◽

Antigen Binding Site ◽

Highly Active ◽

Viral Surface

Monoclonal antibodies (mAbs) that efficiently neutralize SARS-CoV-2 have been developed at an unprecedented speed. Notwithstanding, there is a vague understanding of the various Ab functions induced beyond antigen binding by the heavy-chain constant domain. To explore the diverse roles of Abs in SARS-CoV-2 immunity, we expressed a SARS-CoV-2 spike protein (SP) binding mAb (H4) in the four IgG subclasses present in human serum (IgG1-4) using glyco-engineered Nicotiana benthamiana plants. All four subclasses, carrying the identical antigen-binding site, were fully assembled in planta and exhibited a largely homogeneous xylose- and fucose-free glycosylation profile. The Ab variants ligated to the SP with an up to fivefold increased binding activity of IgG3. Furthermore, all H4 subtypes were able to neutralize SARS-CoV-2. However, H4-IgG3 exhibited an up to 50-fold superior neutralization potency compared with the other subclasses. Our data point to a strong protective effect of IgG3 Abs in SARS-CoV-2 infection and suggest that superior neutralization might be a consequence of cross-linking the SP on the viral surface. This should be considered in therapy and vaccine development. In addition, we underscore the versatile use of plants for the rapid expression of complex proteins in emergency cases.

Download Full-text

Faculty Opinions recommendation of Broadly neutralizing anti-HIV-1 antibodies require Fc effector functions for in vivo activity.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.718872955.793500005 ◽

2014 ◽

Author(s):

Yufei Wang

Keyword(s):

Effector Functions ◽

Anti Hiv ◽

Hiv 1

Download Full-text

Effects of gp120 Inner Domain (ID2) Immunogen Doses on Elicitation of Anti-HIV-1 Functional Fc-Effector Response to C1/C2 (Cluster A) Epitopes in Mice

Microorganisms ◽

10.3390/microorganisms8101490 ◽

2020 ◽

Vol 8 (10) ◽

pp. 1490

Author(s):

Rebekah Sherburn ◽

William D. Tolbert ◽

Suneetha Gottumukkala ◽

Guillaume Beaudoin-Bussières ◽

Andrés Finzi ◽

...

Keyword(s):

Passive Immunization ◽

Neutralizing Antibody ◽

Antibody Repertoire ◽

Effector Functions ◽

Dosing Strategies ◽

Cluster A ◽

Effector Response ◽

Anti Hiv ◽

Hiv 1

Fc-mediated effector functions of antibodies, including antibody-dependent cytotoxicity (ADCC), have been shown to contribute to vaccine-induced protection from HIV-1 infection, especially those directed against non-neutralizing, CD4 inducible (CD4i) epitopes within the gp120 constant 1 and 2 regions (C1/C2 or Cluster A epitopes). However, recent passive immunization studies have not been able to definitively confirm roles for these antibodies in HIV-1 prevention mostly due to the complications of cross-species Fc–FcR interactions and suboptimal dosing strategies. Here, we use our stabilized gp120 Inner domain (ID2) immunogen that displays the Cluster A epitopes within a minimal structural unit of HIV-1 Env to investigate an immunization protocol that induces a fine-tuned antibody repertoire capable of an effective Fc-effector response. This includes the generation of isotypes and the enhanced antibody specificity known to be vital for maximal Fc-effector activities, while minimizing the induction of isotypes know to be detrimental for these functions. Although our studies were done in in BALB/c mice we conclude that when optimally titrated for the species of interest, ID2 with GLA-SE adjuvant will elicit high titers of antibodies targeting the Cluster A region with potent Fc-mediated effector functions, making it a valuable immunogen candidate for testing an exclusive role of non-neutralizing antibody response in HIV-1 protection in vaccine settings.

Download Full-text

Hiding in plain sight: structure and sequence analysis reveals the importance of the antibody DE loop for antibody-antigen binding

10.1101/2020.02.12.946350 ◽

2020 ◽

Cited By ~ 1

Author(s):

Simon P. Kelow ◽

Jared Adolf-Bryfogle ◽

Roland L. Dunbrack

Keyword(s):

High Throughput ◽

High Throughput Sequencing ◽

Data Bank ◽

Antigen Binding ◽

Sequence Variability ◽

Antigen Binding Site ◽

Sequencing Studies ◽

Variable Domains ◽

Complementarity Determining Regions ◽

Hiv 1

AbstractAntibody variable domains contain “complementarity determining regions” (CDRs), the loops that form the antigen binding site. CDRs1-3 are recognized as the canonical CDRs. However, a fourth loop sits adjacent to CDR1 and CDR2 and joins the D and E strands on the antibody v-type fold. This “DE loop” is usually treated as a framework region, even though mutations in the loop affect the conformation of the CDRs and residues in the DE loop occasionally contact antigen. We analyzed the length, structure, and sequence features of all DE loops in the Protein Data Bank, as well as millions of sequences from HIV-1 infected and naïve patients. We refer to the DE loop as H4 and L4 in the heavy and light chain respectively. Clustering the backbone conformations of the most common length of L4 (6 residues) reveals four conformations: two κ-only clusters, one λ-only cluster, and one mixed κ/λ cluster. The vast majority of H4 loops are length-8 and exist primarily in one conformation; a secondary conformation represents a small fraction of H4-8 structures. H4 sequence variability exceeds that of the antibody framework in naïve human high-throughput sequences, and both L4 and H4 sequence variability from λ and heavy germline sequences exceed that of germline framework regions. Finally, we identified dozens of structures in the PDB with insertions in the DE loop, all related to broadly neutralizing HIV-1 antibodies, as well as antibody sequences from high-throughput sequencing studies of HIV-infected individuals, illuminating a possible role in humoral immunity to HIV-1.

Download Full-text

Structural Comparison of Human Anti-HIV-1 gp120 V3 Monoclonal Antibodies of the Same Gene Usage Induced by Vaccination and Chronic Infection

Journal of Virology ◽

10.1128/jvi.00641-18 ◽

2018 ◽

Vol 92 (18) ◽

Cited By ~ 2

Author(s):

Kun-Wei Chan ◽

Ruimin Pan ◽

Matthew Costa ◽

Miroslaw K. Gorny ◽

Shixia Wang ◽

...

Keyword(s):

Hiv Infection ◽

Chronic Infection ◽

Germ Line ◽

Affinity Maturation ◽

Hiv Vaccine ◽

Structural Basis ◽

Antigen Binding ◽

Gene Usage ◽

Anti Hiv ◽

Hiv 1

ABSTRACTElucidating the structural basis of antibody (Ab) gene usage and affinity maturation of vaccine-induced Abs can inform the design of immunogens for inducing desired Ab responses in HIV vaccine development. Analyses of monoclonal Abs (MAbs) encoded by the same immunoglobulin genes at different stages of maturation can help to elucidate the maturation process. We have analyzed four human anti-V3 MAbs with the same VH1-3*01 and VL3-10*01 gene usage. Two MAbs, TA6 and TA7, were developed from a vaccinee in the HIV vaccine phase I trial DP6-001 with a polyvalent DNA prime/protein boost regimen, and two others, 311-11D and 1334, were developed from HIV-infected patients. The somatic hypermutation (SHM) rates in VH of vaccine-induced MAbs are lower than in chronic HIV infection-induced MAbs, while those in VL are comparable. Crystal structures of the antigen-binding fragments (Fabs) in complex with V3 peptides show that these MAbs bind the V3 epitope with a new cradle-binding mode and that the V3 β-hairpin lies along the antigen-binding groove, which consists of residues from both heavy and light chains. Residues conserved from the germ line sequences form specific binding pockets accommodating conserved structural elements of the V3 crown hairpin, predetermining the Ab gene selection, while somatically mutated residues create additional hydrogen bonds, electrostatic interactions, and van der Waals contacts, correlating with an increased binding affinity. Our data provide a unique example of germ line sequences determining the primordial antigen-binding sites and SHMs correlating with affinity maturation of Abs induced by vaccine and natural HIV infection.IMPORTANCEUnderstanding the structural basis of gene usage and affinity maturation for anti-HIV-1 antibodies may help vaccine design and development. Antibodies targeting the highly immunogenic third variable loop (V3) of HIV-1 gp120 provide a unique opportunity for detailed structural investigations. By comparing the sequences and structures of four anti-V3 MAbs at different stages of affinity maturation but of the same V gene usage, two induced by vaccination and another two by chronic infection, we provide a fine example of how germ line sequence determines the essential elements for epitope recognition and how affinity maturation improves the antibody's recognition of its epitope.

Download Full-text

Enhancement of antibody-dependent cellular cytotoxicity and phagocytosis in anti‐HIV‐1 bovine chimeric broadly-neutralizing antibodies.

Journal of Virology ◽

10.1128/jvi.00219-21 ◽

2021 ◽

Author(s):

Jack M Edwards ◽

Behnaz Heydarchi ◽

Georges Khoury ◽

Natalia A Salazar-Quiroz ◽

Christopher A Gonelli ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Variable Region ◽

Cellular Cytotoxicity ◽

Antibody Dependent Cellular Cytotoxicity ◽

Broadly Neutralizing Antibodies ◽

Effector Functions ◽

Human Igg1 ◽

Topical Microbicides ◽

Anti Hiv ◽

Hiv 1

No prophylactic vaccine has provided robust protection against HIV-1. Vaccine-induced broadly neutralizing antibodies (bnAbs) have not been achieved in humans and most animals, however cows vaccinated with HIV-1 envelope trimers produce bnAbs with unusually long third heavy complementarity determining regions (CDRH3). Alongside neutralization, Fc-mediated effector functions including antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis (ADP) may be critical for in vivo bnAb antiviral activity. Here, we aimed to augment the Fc-dependent effector functions of a chimeric human-bovine bnAb, NC-Cow1, which binds the CD4 binding site (CD4bs) and exhibits broader and more potent neutralization than most human CD4bs bnAbs by using an exceptionally long 60aa CDRH3. The bovine variable region of NC-Cow1 was paired with a human IgG1 Fc region mutated to create three variants: G236R/L328R (GRLR) that abrogates Fc-gamma receptor (FcγR) binding, and two variants that enhance binding: G236A/S239D/I332E (GASDIE) and G236A/S239D/A330L/I332E (GASDALIE). Both GASDIE and GASDALIE improved binding to human FcγRIIA and FcγRIIIA, enhanced human NK cell activation and mediated higher levels of ADCC and ADP activity compared to the wild-type human IgG1 Fc. GASDALIE mediated higher phagocytic activity compared to GASDIE. As expected, GRLR eliminated binding to FcγRs and did not mediate ADCC or ADP. We demonstrated that mutations in the human Fc region of bovine chimeric antibodies with ultra-long CDRH3 regions could enhance antibody effector functions while maintaining envelope binding and neutralization. This study will have significant implications in the development of multifunctional anti-HIV antibodies, which may be important to prevent HIV-1 transmission in an antibody-based topical microbicide. IMPORTANCE Despite successful antiviral chemotherapy, HIV is still a lifelong persistent virus and no vaccine yet prevents HIV transmission. Topical microbicides offer an important alternative method to prevent sexual transmission of HIV-1. With the production of highly potent anti-HIV-1 bnAbs and multifunctional antibodies, monoclonal antibodies are now important prophylactic agents. Recently discovered anti-HIV-1 bovine bnAbs (with higher potency and breadth than most human bnAbs) could be novel candidates as potent topical microbicides. Our study is significant as it demonstrates the compatibility of combining bovine-derived neutralization with human-derived antibody-effector functions. This study is a new approach to antibody engineering that strengthens the feasibility of using high potency bovine variable region bnAbs with augmented Fc function and promotes them as a strong candidate for antibody-mediated therapies.

Download Full-text

Quantifying the contribution of Fc-mediated effector functions to the antiviral activity of anti–HIV-1 IgG1 antibodies in vivo

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2008190117 ◽

2020 ◽

Vol 117 (30) ◽

pp. 18002-18009 ◽

Cited By ~ 3

Author(s):

Pengfei Wang ◽

Mili R. Gajjar ◽

Jian Yu ◽

Neal N. Padte ◽

Agegnehu Gettie ◽

...

Keyword(s):

Viral Load ◽

Antiviral Activity ◽

Viral Infections ◽

Rhesus Macaques ◽

Virus Neutralization ◽

Effector Functions ◽

Relative Contribution ◽

Anti Hiv ◽

Hiv 1

In combating viral infections, the Fab portion of an antibody could mediate virus neutralization, whereas Fc engagement of Fc-γ receptors (FcγRs) could mediate an array of effector functions. Evidence abounds that effector functions are important in controlling infections by influenza, Ebola, or HIV-1 in animal models. However, the relative contribution of virus neutralization versus effector functions to the overall antiviral activity of an antibody remains unknown. To address this fundamental question in immunology, we utilized our knowledge of HIV-1 dynamics to compare the kinetics of the viral load decline (ΔVL) in infected animals given a wild-type (WT) anti–HIV-1 immunoglobulin G1 (IgG1) versus those given a Fc-Null variant of the same antibody. In three independent experiments in HIV-1–infected humanized mice and one pivotal experiment in simian–human immunodeficiency virus (SHIV)-infected rhesus macaques, an earlier and sharper decline in viral load was consistently detected for the WT antibody. Quantifications of the observed differences indicate that Fc-mediated effector functions accounted for 25–45% of the total antiviral activity in these separate experiments. In this study, Fc-mediated effector functions have been quantified in vivo relative to the contribution of virus neutralization mediated by the Fab.

Download Full-text

Cocrystal Structures of Antibody N60-i3 and Antibody JR4 in Complex with gp120 Define More Cluster A Epitopes Involved in Effective Antibody-Dependent Effector Function against HIV-1

Journal of Virology ◽

10.1128/jvi.01232-15 ◽

2015 ◽

Vol 89 (17) ◽

pp. 8840-8854 ◽

Cited By ~ 34

Author(s):

Neelakshi Gohain ◽

William D. Tolbert ◽

Priyamvada Acharya ◽

Lei Yu ◽

Tongyun Liu ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Human Monoclonal Antibody ◽

Antiretroviral Drugs ◽

Effector Function ◽

Effector Functions ◽

Immunodeficiency Virus ◽

Cluster A ◽

Definition Of ◽

Anti Hiv ◽

Hiv 1

ABSTRACTAccumulating evidence indicates a role for Fc receptor (FcR)-mediated effector functions of antibodies, including antibody-dependent cell-mediated cytotoxicity (ADCC), in prevention of human immunodeficiency virus type 1 (HIV-1) acquisition and in postinfection control of viremia. Consequently, an understanding of the molecular basis for Env epitopes that constitute effective ADCC targets is of fundamental interest for humoral anti-HIV-1 immunity and for HIV-1 vaccine design. A substantial portion of FcR effector function of potentially protective anti-HIV-1 antibodies is directed toward nonneutralizing, transitional, CD4-inducible (CD4i) epitopes associated with the gp41-reactive region of gp120 (cluster A epitopes). Our previous studies defined the A32-like epitope within the cluster A region and mapped it to the highly conserved and mobile layers 1 and 2 of the gp120 inner domain within the C1-C2 regions of gp120. Here, we elucidate additional cluster A epitope structures, including an A32-like epitope, recognized by human monoclonal antibody (MAb) N60-i3, and a hybrid A32-C11-like epitope, recognized by rhesus macaque MAb JR4. These studies define for the first time a hybrid A32-C11-like epitope and map it to elements of both the A32-like subregion and the seven-layered β-sheet of the gp41-interactive region of gp120. These studies provide additional evidence that effective antibody-dependent effector function in the cluster A region depends on precise epitope targeting—a combination of epitope footprint and mode of antibody attachment. All together these findings help further an understanding of how cluster A epitopes are targeted by humoral responses.IMPORTANCEHIV/AIDS has claimed the lives of over 30 million people. Although antiretroviral drugs can control viral replication, no vaccine has yet been developed to prevent the spread of the disease. Studies of natural HIV-1 infection, simian immunodeficiency virus (SIV)- or simian-human immunodeficiency virus (SHIV)-infected nonhuman primates (NHPs), and HIV-1-infected humanized mouse models, passive transfer studies in infants born to HIV-infected mothers, and the RV144 clinical trial have linked FcR-mediated effector functions of anti-HIV-1 antibodies with postinfection control of viremia and/or blocking viral acquisition. With this report we provide additional definition of the molecular determinants for Env antigen engagement which lead to effective antibody-dependent effector function directed to the nonneutralizing CD4-dependent epitopes in the gp41-reactive region of gp120. These findings have important implications for the development of an effective HIV-1 vaccine.

Download Full-text