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Plant Biotechnology Journal Aug 2019The discovery of broadly neutralizing antibodies (bNAbs) has been a major step towards better prophylactic and therapeutic agents against human immunodeficiency virus...
The discovery of broadly neutralizing antibodies (bNAbs) has been a major step towards better prophylactic and therapeutic agents against human immunodeficiency virus type 1 (HIV-1). However, effective therapy will likely require a combination of anti-HIV agents to avoid viral evasion. One possible solution to this problem is the creation of bispecific molecules that can concurrently target two vulnerable sites providing synergistic inhibitory effects. Here, we describe the production in plants and anti-HIV activity of a novel bispecific fusion protein consisting of the antigen-binding fragment (Fab) of the CD4 binding site-specific bNAb VRC01 and the antiviral lectin Avaren, which targets the glycan shield of the HIV-1 envelope (VRC01 -Avaren). This combination was justified by a preliminary experiment demonstrating the synergistic HIV-1 neutralization activity of VRC01 and Fc-fused Avaren dimer (Avaren-Fc). Using the GENEWARE tobacco mosaic virus vector, VRC01 -Avaren was expressed in Nicotiana benthamiana and purified using a three-step chromatography procedure. Surface plasmon resonance and ELISA demonstrated that both the Avaren and VRC01 moieties retain their individual binding specificities. VRC01 -Avaren demonstrated enhanced neutralizing activity against representative HIV-1 strains from A, B and C clades, compared to equimolar combinations of VRC01 and Avaren. Notably, VRC01 -Avaren showed significantly stronger neutralizing effects than the bivalent parent molecules VRC01 IgG and Avaren-Fc, with IC values ranging from 48 to 310 pm. These results support the continued development of bispecific anti-HIV proteins based on Avaren and bNAbs, to which plant-based transient overexpression systems will provide an efficient protein engineering and production platform.
Topics: Antibodies, Bispecific; Antibodies, Neutralizing; HIV Antibodies; HIV-1; Lectins; Protein Engineering; Recombinant Fusion Proteins; Nicotiana
PubMed: 30729651
DOI: 10.1111/pbi.13090 -
PloS One 2012Generation of potent anti-HIV antibody responses in mucosal compartments is a potential requirement of a transmission-blocking HIV vaccine. HIV-specific, functional...
BACKGROUND
Generation of potent anti-HIV antibody responses in mucosal compartments is a potential requirement of a transmission-blocking HIV vaccine. HIV-specific, functional antibody responses are present in breast milk, and these mucosal antibody responses may play a role in protection of the majority of HIV-exposed, breastfeeding infants. Therefore, characterization of HIV-specific antibodies produced by B cells in milk could guide the development of vaccines that elicit protective mucosal antibody responses.
METHODS
We isolated B cells from colostrum of an HIV-infected lactating woman with a detectable neutralization response in milk and recombinantly produced and characterized the resulting HIV-1 Envelope (Env)-specific monoclonal antibodies (mAbs).
RESULTS
The identified HIV-1 Env-specific colostrum mAbs, CH07 and CH08, represent two of the first mucosally-derived anti-HIV antibodies yet to be reported. Colostrum mAb CH07 is a highly-autoreactive, weakly-neutralizing gp140-specific mAb that binds to linear epitopes in the gp120 C5 region and gp41 fusion domain. In contrast, colostrum mAb CH08 is a nonpolyreactive CD4-inducible (CD4i) gp120-specific mAb with moderate breadth of neutralization.
CONCLUSIONS
These novel HIV-neutralizing mAbs isolated from a mucosal compartment provide insight into the ability of mucosal B cell populations to produce functional anti-HIV antibodies that may contribute to protection against virus acquisition at mucosal surfaces.
Topics: Antibodies, Monoclonal; Antibodies, Neutralizing; B-Lymphocytes; Colostrum; Epitopes, B-Lymphocyte; Female; Fluorescent Antibody Technique, Indirect; HIV Antibodies; HIV Envelope Protein gp120; Humans; Neutralization Tests; Pregnancy
PubMed: 22624058
DOI: 10.1371/journal.pone.0037648 -
Frontiers in Immunology 2021Despite the discovery that the human immunodeficiency virus 1 (HIV-1) is the pathogen of acquired immunodeficiency syndrome (AIDS) in 1983, there is still no effective...
Despite the discovery that the human immunodeficiency virus 1 (HIV-1) is the pathogen of acquired immunodeficiency syndrome (AIDS) in 1983, there is still no effective anti-HIV-1 vaccine. The major obstacle to the development of HIV-1 vaccine is the extreme diversity of viral genome sequences. Nonetheless, a number of broadly neutralizing antibodies (bNAbs) against HIV-1 have been made and identified in this area. Novel strategies based on using these bNAbs as an efficacious preventive and/or therapeutic intervention have been applied in clinical. In this review, we summarize the recent development of bNAbs and its application in HIV-1 acquisition prevention as well as discuss the innovative approaches being used to try to convey protection within individuals at risk and being treated for HIV-1 infection.
Topics: AIDS Vaccines; Animals; Antibody Specificity; Broadly Neutralizing Antibodies; Gene Transfer Techniques; Genes, env; Genetic Therapy; Genetic Variation; HIV Antibodies; HIV Infections; HIV-1; Humans; Immunity, Humoral; Immunization, Passive; Models, Immunological; Vaccine Development; env Gene Products, Human Immunodeficiency Virus
PubMed: 34354709
DOI: 10.3389/fimmu.2021.697683 -
Cell Reports Feb 2020Approximately 50% of the mass of the Envelope (Env) glycoprotein surface subunit (gp120) of human immunodeficiency virus type 1 (HIV-1) is composed of N-linked...
Approximately 50% of the mass of the Envelope (Env) glycoprotein surface subunit (gp120) of human immunodeficiency virus type 1 (HIV-1) is composed of N-linked carbohydrate. Until now, the dogma has been that HIV-1 lacks O-linked carbohydrate on Env. Here we show that a subset of patient-derived HIV-1 isolates contain O-linked carbohydrate on the variable 1 (V1) domain of Env gp120. We demonstrate the presence of this O-glycosylation both on virions and on gp120 expressed as a secreted protein. Further, we establish that these O-linked glycans can confer a more than 1,000-fold decrease in neutralization sensitivity (IC) to V3-glycan broadly neutralizing antibodies. These findings uncover a structural modification to the HIV-1 Env and suggest a functional role in promoting viral escape from one category of broadly neutralizing antibodies.
Topics: Broadly Neutralizing Antibodies; HIV Antibodies; HIV-1; Humans
PubMed: 32049016
DOI: 10.1016/j.celrep.2020.01.056 -
Journal of Clinical Virology : the... Jun 2017The flow-through INSTI™ HIV-1/HIV-2 Rapid Antibody (INSTI) test is a 60s FDA-approved test for HIV-1 and HIV-2 antibody testing using whole blood and plasma.
BACKGROUND
The flow-through INSTI™ HIV-1/HIV-2 Rapid Antibody (INSTI) test is a 60s FDA-approved test for HIV-1 and HIV-2 antibody testing using whole blood and plasma.
OBJECTIVE
We evaluated the performance of INSTI using plasma and simulated whole blood specimens.
STUDY DESIGN
INSTI's performance in plasma specimens from commercial seroconversion panels was assessed by estimating the relative sensitivity using a 50% cumulative frequency analysis and by comparing its performance with other FDA-approved rapid tests (RTs). INSTI was further evaluated using 320 HIV-1 plasma specimens collected during a cross-sectional study and with 107 HIV-1 and 24 HIV-2 simulated whole blood specimens. Sensitivity and specificity were calculated using 615 known HIV-1 group M/O and 80 HIV-2 (Western blot (WB)-positive), and 497 HIV-negative plasma specimens, respectively.
RESULTS
In HIV-1 seroconversion panels, INSTI became reactive 9days before a positive WB. When compared to FDA-approved antibody-based lateral flow RTs, INSTI detected significantly more early infections. Among HIV-1-infected cross-sectional plasma samples, INSTI detected 23 (27%) of 85 Architect-positive/Multispot-negative or indeterminate specimens. For plasma specimens, the sensitivity was 99.84% for HIV-1 and 100% for HIV-2, and the specificity was 99.80%. Using simulated whole blood from seroconverters, INSTI performed similarly to plasma.
CONCLUSIONS
INSTI performed significantly better than antibody-based lateral flow RTs during early stages of seroconversion. Sensitivity and specificity were within the manufacturer's reported ranges. Considering the observed test performance and the almost immediate results, INSTI is an accurate option to detect HIV-1/HIV-2 antibodies in point-of-care settings where lab testing is not feasible.
Topics: AIDS Serodiagnosis; Algorithms; Blotting, Western; Cross-Sectional Studies; HIV Antibodies; HIV Infections; HIV-1; HIV-2; Humans; Mass Screening; Point-of-Care Systems; Sensitivity and Specificity
PubMed: 28372890
DOI: 10.1016/j.jcv.2017.03.012 -
Medecine Sciences : M/S Jan 2014Sexual transmission is currently the major route of HIV infection worldwide. Neutralizing antibodies (IgG) have demonstrated their role in the protection from... (Review)
Review
Sexual transmission is currently the major route of HIV infection worldwide. Neutralizing antibodies (IgG) have demonstrated their role in the protection from experimental challenge in non-human primate's model. However, these types of antibodies display very specific characteristics and are extremely difficult to induce. Interestingly, antibodies devoid of neutralizing activity have demonstrated additional inhibitory mechanisms dependant of their binding to Fc receptors expressed on antigen presenting cells. These cells may play decisive role at early sexual transmission as they have been proposed to be the first HIV target at the mucosal site. Data from in vivo studies and recent findings following clinical assays demonstrated the importance of these Fc-mediated antibodies dependant mechanism in protection against HIV. Therefore new vaccination strategies including the induction of such type of activities, in addition to neutralizing antibodies, should be developed.
Topics: AIDS Vaccines; Animals; Antibodies, Neutralizing; Antiviral Agents; HIV Antibodies; HIV Infections; Humans; Immunity, Humoral; Immunoglobulin A
PubMed: 24472462
DOI: 10.1051/medsci/20143001016 -
Viruses Mar 2024Efforts to develop vaccine and immunotherapeutic countermeasures against the COVID-19 pandemic focus on targeting the trimeric spike (S) proteins of SARS-CoV-2. Vaccines...
Efforts to develop vaccine and immunotherapeutic countermeasures against the COVID-19 pandemic focus on targeting the trimeric spike (S) proteins of SARS-CoV-2. Vaccines and therapeutic design strategies must impart the characteristics of virion S from historical and emerging variants onto practical constructs such as soluble, stabilized trimers. The virus spike is a heterotrimer of two subunits: S1, which includes the receptor binding domain (RBD) that binds the cell surface receptor ACE2, and S2, which mediates membrane fusion. Previous studies suggest that the antigenic, structural, and functional characteristics of virion S may differ from current soluble surrogates. For example, it was reported that certain anti-glycan, HIV-1 neutralizing monoclonal antibodies bind soluble SARS-CoV-2 S but do not neutralize SARS-CoV-2 virions. In this study, we used single-molecule fluorescence correlation spectroscopy (FCS) under physiologically relevant conditions to examine the reactivity of broadly neutralizing and non-neutralizing anti-S human monoclonal antibodies (mAbs) isolated in 2020. Binding efficiency was assessed by FCS with soluble S trimers, pseudoviruses and inactivated wild-type virions representing variants emerging from 2020 to date. Anti-glycan mAbs were tested and compared. We find that both anti-S specific and anti-glycan mAbs exhibit variable but efficient binding to a range of stabilized, soluble trimers. Across mAbs, the efficiencies of soluble S binding were positively correlated with reactivity against inactivated virions but not pseudoviruses. Binding efficiencies with pseudoviruses were generally lower than with soluble S or inactivated virions. Among neutralizing mAbs, potency did not correlate with binding efficiencies on any target. No neutralizing activity was detected with anti-glycan antibodies. Notably, the virion S released from membranes by detergent treatment gained more efficient reactivity with anti-glycan, HIV-neutralizing antibodies but lost reactivity with all anti-S mAbs. Collectively, the FCS binding data suggest that virion surfaces present appreciable amounts of both functional and nonfunctional trimers, with neutralizing anti-S favoring the former structures and non-neutralizing anti-glycan mAbs binding the latter. S released from solubilized virions represents a nonfunctional structure bound by anti-glycan mAbs, while engineered soluble trimers present a composite structure that is broadly reactive with both mAb types. The detection of disparate antigenicity and immunoreactivity profiles in engineered and virion-associated S highlight the value of single-virus analyses in designing future antiviral strategies against SARS-CoV-2.
Topics: Humans; Spike Glycoprotein, Coronavirus; SARS-CoV-2; Pandemics; COVID-19; Antibodies, Neutralizing; HIV Antibodies; Antibodies, Monoclonal; HIV-1; Virion; Antibodies, Viral
PubMed: 38543772
DOI: 10.3390/v16030407 -
Communications Biology Mar 2022The early humoral immune response to acute HIV-1 infection is largely non-neutralizing. The principal target of these antibodies is the primary immunodominant region...
The early humoral immune response to acute HIV-1 infection is largely non-neutralizing. The principal target of these antibodies is the primary immunodominant region (PID) on the gp41 fusion protein. The PID is a highly conserved 15-residue region displayed on the surface of HIV-1 virions. In this study, we analyzed the humoral determinants of HIV-1 gp41 PID binding using biophysical, structural, and computational methods. In complex with a patient-derived near-germline antibody fragment, the PID motif adopts an elongated random coil, whereas the PID bound to affinity-matured Fab adopts a strand-turn-helix conformation. Molecular dynamics simulations showed that the PID is structurally plastic suggesting that the PID can form an ensemble of structural states recognized by various non-neutralizing antibodies, facilitating HIV-1 immunodominance observed in acute and chronic HIV-1 infections. An improved understanding of how the HIV-1 gp41 PID misdirects the early humoral response should guide the development of an effective HIV-1 vaccine.
Topics: HIV Antibodies; HIV Envelope Protein gp41; HIV-1; Humans; Immunodominant Epitopes; Protein Conformation
PubMed: 35361878
DOI: 10.1038/s42003-022-03235-w -
Immunological Reviews Nov 2012Human immunodeficiency virus-1 (HIV-1) envelope protein (Env) and influenza hemagglutinin (HA) are the surface glycoproteins responsible for viral entry into host cells,... (Review)
Review
Human immunodeficiency virus-1 (HIV-1) envelope protein (Env) and influenza hemagglutinin (HA) are the surface glycoproteins responsible for viral entry into host cells, the first step in the virus life cycle necessary to initiate infection. These glycoproteins exhibit a high degree of sequence variability and glycosylation, which are used as strategies to escape host immune responses. Nonetheless, antibodies with broadly neutralizing activity against these viruses have been isolated that have managed to overcome these barriers. Here, we review recent advances in the structural characterization of these antibodies with their viral antigens that defines a few sites of vulnerability on these viral spikes. These broadly neutralizing antibodies tend to focus their recognition on the sites of similar function between the two viruses: the receptor-binding site and membrane fusion machinery. However, some sites of recognition are unique to the virus neutralized, such as the dense shield of oligomannose carbohydrates on HIV-1 Env. These observations are discussed in the context of structure-based design strategies to aid in vaccine design or development of antivirals.
Topics: AIDS Vaccines; Antibodies, Neutralizing; Antigenic Variation; Gene Products, env; HIV Antibodies; HIV-1; Hemagglutinin Glycoproteins, Influenza Virus; Humans; Influenza A virus; Influenza Vaccines; Models, Molecular; Receptors, Virus; Species Specificity; Structural Homology, Protein; Virus Internalization
PubMed: 23046130
DOI: 10.1111/imr.12005 -
Retrovirology Jun 2021HIV remains one of the most important health issues worldwide, with almost 40 million people living with HIV. Although patients develop antibodies against the virus, its...
BACKGROUND
HIV remains one of the most important health issues worldwide, with almost 40 million people living with HIV. Although patients develop antibodies against the virus, its high mutation rate allows evasion of immune responses. Some patients, however, produce antibodies that are able to bind to, and neutralise different strains of HIV. One such 'broadly neutralising' antibody is 'N6'. Identified in 2016, N6 can neutralise 98% of HIV-1 isolates with a median IC of 0.066 µg/mL. This neutralisation breadth makes N6 a very promising therapeutic candidate.
RESULTS
N6 was expressed in a glycoengineered line of N. benthamiana plants (pN6) and compared to the mammalian cell-expressed equivalent (mN6). Expression at 49 mg/kg (fresh leaf tissue) was achieved in plants, although extraction and purification are more challenging than for most plant-expressed antibodies. N-glycoanalysis demonstrated the absence of xylosylation and a reduction in α(1,3)-fucosylation that are typically found in plant glycoproteins. The N6 light chain contains a potential N-glycosylation site, which was modified and displayed more α(1,3)-fucose than the heavy chain. The binding kinetics of pN6 and mN6, measured by surface plasmon resonance, were similar for HIV gp120. pN6 had a tenfold higher affinity for FcγRIIIa, which was reflected in an antibody-dependent cellular cytotoxicity assay, where pN6 induced a more potent response from effector cells than that of mN6. pN6 demonstrated the same potency and breadth of neutralisation as mN6, against a panel of HIV strains.
CONCLUSIONS
The successful expression of N6 in tobacco supports the prospect of developing a low-cost, low-tech production platform for a monoclonal antibody cocktail to control HIV in low-to middle income countries.
Topics: Antibodies, Neutralizing; HEK293 Cells; HIV Antibodies; HIV Infections; HIV-1; Humans; Inhibitory Concentration 50; Neutralization Tests; Plant Leaves; Nicotiana
PubMed: 34183026
DOI: 10.1186/s12977-021-00560-6