Anti-gp120 Antibodies Research Articles

P-D7 Structural mimicry of the antigen binding modes of rhesus macaque and human anti-gp120 V3 antibodies

Background: Nonhuman primates (NHPs), such as rhesus macaques, are currently the best preclinical models for studying HIV vaccines. The details of how NHP Abs can mimic human Abs will be useful in evaluating NHP Ab responses to HIV vaccine candidates. The extensive knowledge of the antigen binding modes of anti-V3 Abs allows a structural comparison of NHP and human Ab binding modes. Methods: We have crystallized and determined the epitope complex structures of rhesus macaque anti-V3 monoclonal Abs (mAbs) 2.10A (encoded by a VH5 family gene and derived from an animal infected with SHIV SF162P4) and P2A10 and P2E3 (encoded by VH3 family genes and from an animal infected with SHIV SF162P3N). These structures were compared to the Ab-epitope interactions of human anti-V3 mAbs encoded by the equivalent human genes. Results: Human anti-V3 mAbs preferentially use VH5, VH1 and VH3 family genes with the antigen binding sites resembling either a cradle or ladle, respectively, as previously described. Similarly, rhesus macaque mAb 2.10A utilizes the cradle-binding mode while mAbs P2A10 and P2E3 use the ladle-binding mode. Additionally, the amino acids encoded by both human and macaque VH3 and VH5 family germline genes are critical for shaping the antigen binding sites. Thus, key elements in the antigen-Ab interactions are preserved between human and rhesus macaque mAbs. Conclusions: The antigen binding modes of human and rhesus macaque mAbs targeting HIV-1 gp120 V3 epitopes were found to be structurally extremely similar at the atomic level.

Effects of Anti-Glycoprotein Antibodies on Response of Immune Thrombocytopenia Patients to Thrombopoietin Receptor Agonists and on Megakaryocytes Viability

BackgroundImmune Thrombocytopenia (ITP) is a bleeding disorder due to a combination of increased platelet destruction and reduced production, often secondary to anti-platelet/megakaryocyte antibodies.The presence of antibodies to glycoproteins (GP) IIb/IIIa (integrin αIIbβ3) and GPIb/IX, detected in majority of ITP patients, may correspond to different responses to treatment, i.e., anti-GPIb is associated with more severe disease, and less responsive to intravenous immunoglobulins and steroids.Thrombopoietin Receptor Agonists (TPO-RA) increase platelet production by stimulation of megakaryopoesis. Predictors of response to TPO-RA and influence of antibody profile on response are currently unknown. In our previous study we investigated Absolute Immature Platelet Fraction (A-IPF) prior to TPO-RA treatment and did not find a correlation between A-IPF, anti-GP antibodies, and platelet counts.The aims of this study were to further investigate:1. The role of anti-GP antibodies in response to TPO-RA;2. Effect of patients' antibodies on megakaryocyte (MK) viability, maturation, apoptosis and formation of proplatelets (in vitro);3. The influence of patients' clinical characteristics on response to TPO-RA.Materials and Methods91 patients with persistent or chronic ITP, were treated at Weill Medical College of Cornell University until January 2015 with TPO-RAs: 52 patients received eltrombopag, 22 romiplostim and 17 avatrombopag. Serum samples of 84 patients were analyzed for the presence of anti-GP by MAIPA assay as previously described.Patients with baseline platelet counts less than <30x109/L were defined as responders to TPO-RA if the average of their six median monthly platelet counts was ≥50x109/L and doubled from average baseline counts (prior to TPO-RA). Patients with baseline platelet counts 30-50x109/L were responders if the average platelet count was ≥75x109/L.MKs were derived from human umbilical cord blood stem cells as previously described. Cells were grown using SFEM medium, adding on day 0 of culture 50 ng/ml recombinant TPO and aliquots of serum of ITP patients or healthy controls. The percentages of immature (CD41+/CD42-), mature (CD41+/CD42+), viable and apoptotic MKs were analyzed by flow cytometry on day 12. Apoptosis was analyzed by measuring Mitochondrial Outer Membane Potential (MOMP) and Phosphatidyl Serine (PS) externalization. MKs were considered apoptotic if they had positive staining for PS externalization, viable if positive for MOMP, and dead if positive for 7-Aminoactinomycin D (7AAD).Proplatelet formation by MKs was analyzed by microscopy.Statistical analysis using unpaired T-test and Pearson correlation test were performed.ResultsNinety-one patients were included, 40 male (44%) and 51 female (56%), with a median age of 37.4 years (range 2-87). Median duration of ITP before TPO-RA treatment was 8 years (range 0.3-45).The 18/91 (19.8%) non-responders to TPO-RA were not different from the 73/91 responders in age, gender, number of prior treatments, duration of ITP, and past splenectomy. The presence of either or both anti-GP antibodies was correlated with average lower platelet counts on TPO-RA: 82.3 x109/L versus 123x109/L in patients without detected antibodies ("neither") (p=0.003). However, the response to TPO-RA was not influenced by the type of antibody: in patients with anti-GPIb the average platelet count was 76.1x109/L, and with anti-GPIIb/IIIa 80.7x109/L (Figure 1). In culture, excess dead MKs were found in anti-GPIb group and antiGPIb&antiGPIIb/IIIa group compared to "neither" group (p=0.0013 and p=0.027 respectively) and comparing antiGPIb&antiGPIIb/IIIa to control (p=0.0025). We did not observe changes in the degree of MK apoptisis or in MK maturation in the presence of serum antibodies. In cultures treated with serum of patients having anti-GPIb, less proplatelets were detected comparing to control (p=0.044) or to "neither" (p=0.0039).We conclude that patients with anti-GP antibodies respond less to TPO-RA, however there is no difference in response to TPO-RA between patients having anti-GPIb and anti-GPIIb/IIIa, unlike responses to other treatment modalities (e.g., steroids or immunoglobulins). TPO-RA could be a preferable treatment option in ITP patients having anti-GPIb. [Display omitted] DisclosuresOff Label Use: Eltrombopag, romiplostim and avatrombopag are a thrombopoietin receptor agonist approved for the treatment of thrombocytopenia in adults with chronic ITP. In some preliminary studies these medicines found as safe and effective treatment option in children and adolescents. Bussel:amgen: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding; GlaxoSmithKline: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding; Cangene: Research Funding; Novartis: Consultancy, Research Funding; Genzyme: Consultancy; BiologicTx: Research Funding; Ligand: Consultancy, Research Funding; Eisai: Consultancy, Research Funding; Shionogi: Consultancy, Research Funding; momenta: Consultancy; Protalex: Consultancy; Symphogen: Consultancy.

Antibody Derived Peptides for Detection of Ebola Virus Glycoprotein.

BackgroundCurrent Ebola virus (EBOV) detection methods are costly and impractical for epidemic scenarios. Different immune-based assays have been reported for the detection and quantification of Ebola virus (EBOV) proteins. In particular, several monoclonal antibodies (mAbs) have been described that bind the capsid glycoprotein (GP) of EBOV GP. However, the currently available platforms for the design and production of full-length mAbs are cumbersome and costly. The use of antibody fragments, rather than full-length antibodies, might represent a cost-effective alternative for the development of diagnostic and possibly even therapeutic alternatives for EBOV.Methods/Principal FindingsWe report the design and expression of three recombinant anti-GP mAb fragments in Escherichia coli cultures. These fragments contained the heavy and light variable portions of the three well-studied anti-GP full-length mAbs 13C6, 13F6, and KZ52, and are consequently named scFv-13C6, scFv-13F6, and Fab-KZ52, respectively. All three fragments exhibited specific anti-GP binding activity in ELISA experiments comparable to that of full-length anti-GP antibodies (i.e., the same order of magnitude) and they are easily and economically produced in bacterial cultures.Conclusion/SignificanceAntibody fragments might represent a useful, effective, and low cost alternative to full-length antibodies in Ebola related capture and diagnostics applications.

Interferon α/β Receptor-Deficient Mice as a Model for Ebola Virus Disease.

A major obstacle in ebolavirus research is the lack of a small-animal model for Sudan virus (SUDV), as well as other wild-type (WT) ebolaviruses. Here, we expand on research by Bray and by Lever et al suggesting that WT ebolaviruses are pathogenic in mice deficient for the type 1 interferon (IFN) α/β receptor (IFNα/βR-/-). We examined the disease course of several WT ebolaviruses: Boneface (SUDV/Bon) and Gulu variants of SUDV, Ebola virus (EBOV), Bundibugyo virus (BDBV), Taï Forest virus, and Reston virus (RESTV). We determined that exposure to WT SUDV or EBOV results in reproducible signs of disease in IFNα/βR-/- mice, as measured by weight loss and partial lethality. Vaccination with the SUDV or EBOV glycoprotein (GP)-expressing Venezuelan equine encephalitis viral replicon particle vaccine protected these mice from SUDV/Bon and EBOV challenge, respectively. Treatment with SUDV- or EBOV-specific anti-GP antibodies protected mice from challenge when delivered 1-3 days after infection. Serial sampling experiments revealed evidence of disseminated intravascular coagulation in the livers of mice infected with the Boneface variant of SUDV, EBOV, and BDBV. Taken together, these data solidify the IFNα/βR-/- mouse as an important and useful model for the study of WT EBOV disease.

Characterization of Immune Responses Induced by Ebola Virus Glycoprotein (GP) and Truncated GP Isoform DNA Vaccines and Protection Against Lethal Ebola Virus Challenge in Mice.

In addition to its surface glycoprotein (GP), Ebola virus directs the production of large quantities of a truncated glycoprotein isoform (sGP) that is secreted into the extracellular space. We recently reported that sGP actively diverts host antibody responses against the epitopes that it shares with GP and thereby allows itself to absorb anti-GP antibodies, a phenomenon we termed "antigenic subversion." To investigate the effect of antigenic subversion by sGP on protection against virus infection, we compared immune responses induced by different prime-boost immunization regimens with GP and sGP DNA vaccines in mice and their efficacy against lethal Ebola virus challenge. Similar levels of anti-GP antibodies were induced by 2 immunizations with sGP and GP DNA vaccines. However, 2 immunizations with GP but not sGP DNA vaccine fully protected mice from lethal challenge. Boosting with sGP or GP DNA vaccine in mice that had been primed by GP or sGP DNA vaccine augmented the levels of anti-GP antibody responses and further improved protective efficacy against Ebola virus infection. These results show that both the quality and the levels of anti-GP antibody responses affect the efficacy of protection against Ebola virus infection.

Azasugar-containing phosphorothioate oligonucleotide (AZPSON) DBM-2198 inhibits human immunodeficiency virus type 1 (HIV-1) replication by blocking HIV-1 gp120 without affecting the V3 region.

DBM-2198, a six-membered azasugar nucleotide (6-AZN)-containing phosphorothioate (P = S) oligonucleotide (AZPSON), was described in our previous publication [Lee et al. (2005)] with regard to its antiviral activity against a broad spectrum of HIV-1 variants. This report describes the mechanisms underlying the anti-HIV-1 properties of DBM-2198. The LTR-mediated reporter assay indicated that the anti-HIV-1 activity of DBM-2198 is attributed to an extracellular mode of action rather than intracellular sequence-specific antisense activity. Nevertheless, the antiviral properties of DBM-2198 and other AZPSONs were highly restricted to HIV-1. Unlike other P = S oligonucleo-tides, DBM-2198 caused no host cell activation upon administration to cultures. HIV-1 that was pre-incubated with DBM-2198 did not show any infectivity towards host cells whereas host cells pre-incubated with DBM-2198 remained susceptible to HIV-1 infection, suggesting that DBM-2198 acts on the virus particle rather than cell surface molecules in the inhibition of HIV-1 infection. Competition assays for binding to HIV-1 envelope protein with anti-gp120 and anti-V3 antibodies revealed that DBM-2198 acts on the viral attachment site of HIV-1 gp120, but not on the V3 region. This report provides a better understanding of the antiviral mechanism of DBM-2198 and may contribute to the development of a potential therapeutic drug against a broad spectrum of HIV-1 variants.

HIV gp41-Antibody Interaction at the Viral Membrane Interface Defined by EPR Spectroscopy

HIV enters human T cells through the fusion of viral and host-cell membranes. This fusion process is mediated by a surface protein, gp41, and the platform provided by the cholesterol-rich viral membrane. The membrane proximal ectodomain region (MPER) of gp41 plays a critical role in this fusion process and is a major target of anti-gp41 antibodies and vaccine design. Here, EPR and NMR techniques were used to define MPER structure on the membrane, and how neutralizing anti-gp41 antibodies recognize their membrane-immersed epitopes and disrupt a hinge-related function of the MPER. The analyses of several HIV-1 clade B and clade C MPERs revealed a structurally conserved pair of helices immersed in the viral membrane separated by a flexible hinge, which include critical helix capping residues. Double alanine mutations of the capping residues result in an altered hinge structure with a deeper lipid-buried MPER middle region, as well as reduced viral fusion and infectivity. Furthermore, neutralizing anti-gp41 antibodies disrupt the MPER hinge function by perturbing MPER hinge orientation, and/or extracting part of the MPER from the membrane. The interaction can be a stepwise rearrangement through an apparent scoop-like movement of the antibodies’ long and unique CDRH3 segments. Mutations of the CDRH3 segments reduced the ability of the antibodies to extract MPER residues from the membrane, without affecting peptide binding in solution. In addition, MPER-membrane interaction and antibody binding are modulated by lipid composition and cholesterol content. These findings have revealed important features of gp41-antibody interaction at the viral membrane interface.

Topical Mucosal Vaccination with Angiotensin (1-7) and Feline Immunodeficiency Virus Induces Secretory IgA Responses

Background: Pre-existing mucosal generated secretory IgA antibodies may prevent transmission of HIV. The present study aimed to characterize mucosal antibodies generated following topical vaccination with a novel mucosal adjuvant, angiotensin (1-7), in combination with a killed feline immunodeficiency virus (FIV) vaccine used as a model antigen. Methods: Female outbred cats were vaccinated with Fel-O-Vax FIV vaccine agent combined with increasing concentrations of angiotensin (1-7) [A (1-7)] applied topically to oral, vaginal and rectal surfaces weekly for six weeks. Control animals received intramuscular vaccinations with the Fel-O-Vax FIV alone or with A (1-7). Mucosal secretions were evaluated for antibody responses against FIV-p24 antigen or HIV-gp120 antigen. Results: Topical application of whole killed FIV virus with A (1-7) induced substantial secretory IgA (SIgA)-antigp120 antibodies in oral, vaginal and rectal secretions across a wide ranges of A (1-7) dose levels. Intramuscular vaccination of FIV antigen with A (1-7) induced high levels of SIgA-anti-gp120 antibodies at vaginal and rectal sites. The topical application vaccination strategy elicited only very weak systemic immune responses. Conclusion: Angiotensin (1-7) is a potent mucosal vaccine adjuvant.

HIV Gp120 Epitope Profile on Cell-Bound Virions Assessed using Various Microscopy Techniques

Background: HIV gp120 experiences multiple molecular interactions and structural rearrangements during host cell attachment and entry. However, the antigenic correlates of these dynamic changes are essentially unknown for single HIV particles bound to target cells. Gp120 on cell-bound virions is a target for multiple humoral effector mechanisms mediated by anti-gp120 antibodies, including direct neutralizing activity and elimination by Fc-mediated effector functions such as antibody-dependent cell cytotoxicity (ADCC). It is therefore critical to understand the antigenicity of these conserved epitopes that become exposed during productive viral replication, to provide important guidance for designing antiviral strategies such as vaccines to raise protective antibody responses. Methods: We studied the antigenicity of conserved epitopes by visualizing their exposure on single HIV particles as they interact with target TZM-bl cells using confocal microscopy. Epitope exposure was probed by visualizing the binding of cognate antibodies. We then employed direct stochastic optical reconstruction microscopy (dSTORM) to determine the number of antibodies bound to cell-attached HIV, and their location relative to the virus - cell contact site. Results: CD4-induced (CD4i) epitopes thought to be sterically occluded from the virus - cell interface, were exposed on cell-bound HIV gp120. The level of A32, 17b and C11 CD4i antibody staining was similar to neutralizing antibodies b12 and 2G12. The location of these CD4i epitopes was distal to the virus - cell contact site. Conclusion: The patterns of cell-bound gp120 epitope exposure are consistent with the ADCC activities of cognate antibodies. CD4i epitopes were unexpectedly exposed distal to the HIV - cell interface, where they can be accessed by antibodies involved in ADCC. These findings indicate that HIV-1 exhibits a diversity of epitope exposure upon attachment that may provide unique insights for understanding how humoral immunity impacts HIV infection.

Barcoded microchips for biomolecular assays.

Multiplexed assay of analytes is of great importance for clinical diagnostics and other analytical applications. Barcode-based bioassays with the ability to encode and decode may realize this goal in a straightforward and consistent manner. We present here a microfluidic barcoded chip containing several sets of microchannels with different widths, imitating the commonly used barcode. A single barcoded microchip can carry out tens of individual protein/nucleic acid assays (encode) and immediately yield all assay results by a portable barcode reader or a smartphone (decode). The applicability of a barcoded microchip is demonstrated by human immunodeficiency virus (HIV) immunoassays for simultaneous detection of three targets (anti-gp41 antibody, anti-gp120 antibody, and anti-gp36 antibody) from six human serum samples. We can also determine seven pathogen-specific oligonucleotides by a single chip containing both positive and negative controls.

Neutralization-Enhancing RF Antibodies for HIV Vaccines.

Efficient neutralization of HIV is a primary goal both for therapeutic and prophylactic HIV vaccines based on induction of neutralizing antibodies (NAbs) (1, 2). Neutralization capacity of NAbs correlates with their affinity to HIV-1 gp120 envelope glycoprotein (3). In HIV-infected individuals, early IgM antibodies have low affinity for gp120 glycoprotein (3), which is slightly compensated by their pentameric structure and avidity to multivalent structures of the virus. Corresponding IgG1 antibodies (3, 4), which appear later from IgM through the CSR and SHM processes (3, 5), have high affinity and specificity to gp120, but low avidity due to monomeric structure of IgG. The question is how to induce HIVspecific NAbs with both high affinity and avidity to gp120? Anti-HIV IgG antibodies 2F5 and 2G12, switched back to IgM isotype, showed increased avidity and neutralization efficacy (2). Dendritic antibody supramolecules (DAS) in one molecule combine high specificity of IgG with high avidity of IgM (6). It would be of interest to construct such a DAS with IgM as a core, carrying 10 anti-gp120 IgG monomers. Natural monomeric anti-gp120 IgG1 antibodies (3, 4) not only have low avidity insufficient for effective neutralization of HIV but may also contribute to FcR-mediated infection enhancement (7, 8). Does it mean that HIV may use high-affinity IgG antibodies for its own purposes? Fortunately, in nature there exist some helpful network-like regulatory mechanisms with a key role of RF antibodies (9). Rheumatoid factor (RF) is an autoantibody which specifically binds Fc region of IgG (10, 11). Apart from pathologic RF in rheumatoid arthritis (12), natural RF appears in many non-rheumatoid states (13). Possible beneficial physiological roles of RF include enhanced clearance of immune complexes (ICs) (14), amplification of IgG response to pathogens (15), and enhancement of virus neutralization (16, 17). RF can be induced in vivo in a highly specific way either by secondary immunization with protein antigens or in response to immunization with the newly formed ICs (10, 11). New antigenic determinants, which appear in the Fc region of IgG antibody upon antibody-antigen complex formation, may strengthen the specificity of RF (9). The level of RF was significantly higher in some of HIV-infected individuals compared to control groups (18–22). RFs were mainly IgA, IgM immunoglobulins with specificity against anti-HIV IgG (20). RF-mediated enhancement of antiHIV IgG neutralization activity was found in the sera from MCTD patients (17). Authors suggested that RF is promising for passive immunotherapy based on NAbs (17). Can RF play a key role in specific enhancement of IgG-mediated neutralization of HIV in vivo? Measurements of RF level both in long-term non-progressors (23) and dual-infected individuals (24) might give some clues. Repeated immunization of uninfected macaques with HIV1 gp120 glycoprotein may allow researchers not only to track the kinetics of RF induction but also to elucidate whether neutralization-enhancing RF antibodies can protect macaques against subsequent challenge with SHIV. Repeated immunization of SHIV-infected macaques with HIV1 gp120 glycoprotein might show whether neutralization-enhancing RF antibodies can prolong the asymptomatic period and delay the onset of AIDS. The level of RF is not stable and has a tendency to decline during the acute phase of HIV infection (22). Prolonged repeated immunizations with gp120 (e.g., immunizations every 3 weeks; the actual time between immunizations will be adjusted according to measurements of RF level in patients) would be the solution to keep the level of neutralization-enhancing RF antibodies constantly high. Singleadministration vaccine (SAV) technology, which is based on pulsatile release of gp120 from biodegradable polymeric microspheres, mimics repeated immunization scheme, and allows vaccination to be done in one shot (25, 26). Patient-specific therapeutic HIV vaccines (1) even in simplified version, using the only one gp120 variant formed after completion of HIV population homogenization process (27), can be performed via repeated immunizations from biodegradable polymeric microspheres under SAV (25, 26) technology platform. Prophylactic HIV vaccines can be based on the variations of sequential scheme (28) for prolonged pulsatile release of gp120 glycoproteins from biodegradable polymeric microspheres in single-shot (25, 26) way convenient for both patients and doctors. Studies (15, 17) have shown the high potential of neutralization-enhancing RF antibodies, but several principal questions arise:

Atomic force microscopy fishing and mass spectrometry identification of gp120 on immobilized aptamers.

Atomic force microscopy (AFM) was applied to carry out direct and label-free detection of gp120 human immunodeficiency virus type 1 envelope glycoprotein as a target protein. This approach was based on the AFM fishing of gp120 from the analyte solution using anti-gp120 aptamers immobilized on the AFM chip to count gp120/aptamer complexes that were formed on the chip surface. The comparison of image contrasts of fished gp120 against the background of immobilized aptamers and anti-gp120 antibodies on the AFM images was conducted. It was shown that an image contrast of the protein/aptamer complexes was two-fold higher than the contrast of the protein/antibody complexes. Mass spectrometry identification provided an additional confirmation of the target protein presence on the AFM chips after biospecific fishing to avoid any artifacts.

HIV-1 envelope gp41 antibodies can originate from terminal ileum B cells that share cross-reactivity with commensal bacteria.

Monoclonal antibodies derived from blood plasma cells of acute HIV-1-infected individuals are predominantly targeted to the HIV Env gp41 and cross-reactive with commensal bacteria. To understand this phenomenon, we examined anti-HIV responses in ileum B cells using recombinant antibody technology and probed their relationship to commensal bacteria. The dominant ileum B cell response was to Env gp41. Remarkably, a majority (82%) of the ileum anti-gp41 antibodies cross-reacted with commensal bacteria, and of those, 43% showed non-HIV-1 antigen polyreactivity. Pyrosequencing revealed shared HIV-1 antibody clonal lineages between ileum and blood. Mutated immunoglobulin G antibodies cross-reactive with both Env gp41 and microbiota could also be isolated from the ileum of HIV-1 uninfected individuals. Thus, the gp41 commensal bacterial antigen cross-reactive antibodies originate in the intestine, and the gp41 Env response in HIV-1 infection can be derived from a preinfection memory B cell pool triggered by commensal bacteria that cross-react with Env.

Site-selective solid-phase synthesis of a CCR5 sulfopeptide library to interrogate HIV binding and entry.

Tyrosine (Tyr) sulfation is a common post-translational modification that is implicated in a variety of important biological processes, including the fusion and entry of human immunodeficiency virus type-1 (HIV-1). A number of sulfated Tyr (sTyr) residues on the N-terminus of the CCR5 chemokine receptor are involved in a crucial binding interaction with the gp120 HIV-1 envelope glycoprotein. Despite the established importance of these sTyr residues, the exact structural and functional role of this post-translational modification in HIV-1 infection is not fully understood. Detailed biological studies are hindered in part by the difficulty in accessing homogeneous sulfopeptides and sulfoproteins through biological expression and established synthetic techniques. Herein we describe an efficient approach to the synthesis of sulfopeptides bearing discrete sulfation patterns through the divergent, site-selective incorporation of sTyr residues on solid support. By employing three orthogonally protected Tyr building blocks and a solid-phase sulfation protocol, we demonstrate the synthesis of a library of target N-terminal CCR5(2-22) sulfoforms bearing discrete and differential sulfation at Tyr10, Tyr14, and Tyr15, from a single resin-bound intermediate. We demonstrate the importance of distinct sites of Tyr sulfation in binding gp120 through a competitive binding assay between the synthetic CCR5 sulfopeptides and an anti-gp120 monoclonal antibody. These studies revealed a critical role of sulfation at Tyr14 for binding and a possible additional role for sulfation at Tyr10. N-terminal CCR5 variants bearing a sTyr residue at position 14 were also found to complement viral entry into cells expressing an N-terminally truncated CCR5 receptor.

A recombinant adenovirus-based vector elicits a specific humoral immune response against the V3 loop of HIV-1 gp120 in mice through the “Antigen Capsid-Incorporation” strategy

BackgroundDue to potential advantages, human adenoviral vectors have been evaluated pre-clinically as recombinant vaccine vectors against several cancers and infectious diseases, including human immunodeficiency virus (HIV) infection. The V3 loop of HIV-1 glycoprotein 120 (gp120) contains important neutralizing epitopes and plays key roles in HIV entry and infectivity.MethodsIn order to investigate the humoral immune response development against portions of the V3 loop, we sought to generate four versions of adenovirus (Ad)-based V3 vectors by incorporating four different antigen inserts into the hypervariable region 1 (HVR1) of human adenovirus type 5 (hAd5) hexon. The strategy whereby antigens are incorporated within the adenovirus capsid is known as the “Antigen Capsid-Incorporation” strategy.ResultsOf the four recombinant vectors, Ad-HVR1-lgs-His6-V3 and Ad-HVR1-long-V3 had the capability to present heterologous antigens on capsid surface, while maintaining low viral particle to infectious particle (VP/IP) ratios. The VP/IP ratios indicated both high viability and stability of these two vectors, as well as the possibility that V3 epitopes on these two vectors could be presented to immune system. Furthermore, both Ad-HVR1-lgs-His6-V3 and Ad-HVR1-long-V3 could, to some extent escape the neutralization by anti-adenovirus polyclonal antibody (PAb), but rather not the immunity by anti-gp120 (902) monoclonal antibody (MAb). The neutralization assay together with the whole virus enzyme-linked immunosorbent assay (ELISA) suggested that these two vectors could present V3 epitopes similar to the natural V3 presence in native HIV virions. However, subsequent mice immunizations clearly showed that only Ad-HVR1-lgs-His6-V3 elicited strong humoral immune response against V3. Isotype ELISAs identified IgG2a and IgG2b as the dominant IgG isotypes, while IgG1 comprised the minority.ConclusionsOur findings demonstrated that human adenovirus (hAd) vectors which present HIV antigen via the “Antigen Capsid-Incorporation” strategy could successfully elicit antigen-specific humoral immune responses, which could potentially open an avenue for the development of Ad-based HIV V3 vaccines.

B cell subpopulations alteration in pathogenic and nonpathogenic SIV infections (VIR7P.1061)

Abstract SIV/HIV infection is associated with B cell dysfunction, the significance of which is not known. We compared the B cell dynamics in blood, lymph nodes (LNs) and gut during SIVsab pathogenic infection of pigtailed macaques (PTMs) and nonprogressive SIVsab infection of African green monkeys (AGMs) by monitoring the B cell subsets, B regulatory cells (Bregs), CD4+ T follicular helper (Tfh) cells, B cell immune activation, apoptosis, exhaustion and homing to the intestine. Binding anti-gp41 and neutralizing antibodies (Nabs) were also measured. Acute SIV infection induced a transient but significant loss of total circulating B cells in PTMs only. In AGMs, total B cell from LNs increased during chronic infection. B cells transiently increased in the gut and naïve subset decreased in all three compartments in both species. Memory B cell subsets showed a different dynamic for both species. Bregs increased in PTM, correlated with IL-10 expression. B cell proliferation occurred in both species. PTMs expressed high baseline levels of α4β7, high degree of B cell activation and apoptosis and high frequency of Tfh. The dynamics of Ab response was similar in both species, but rapid progressor PTMs lacked the ability to mount anti-gp41. Our data show that B cell dysfunction only occurs in pathogenic SIV infection. While B cell changes were not correlated with production of Nabs, rapid disease progression associated a severe incapacity to mount an anti-SIV Ab response.

Nedd4-Mediated Increase in HIV-1 Gag and Env Proteins and Immunity following DNA-Vaccination of BALB/c Mice

The late assembly domain of many viruses is critical for budding. Within these domains, encoded in viral structural proteins, are the conserved motifs PTAP, PPxY and YPxL. These sequences are the key determinants for association of viral proteins with intracellular molecules such as Tsg101, Nedd4 and AIP1/ALIX. While roles for Tsg101 and AIP1/ALIX in HIV-1 budding have been well established, less is known about the role of Nedd4. Recent studies, however, have identified a function for Nedd4-like protein in HIV-1 release. In this study, we investigated post-transcriptional changes of Nedd4 following SHIVSF162P3 infection of rhesus macaques, its role on HIV-1 p24 and gp120 levels in vitro and its potential as an immune modulator in HIV vaccination of BALB/c mice. Increased Nedd4 protein levels were noted in both CD4+ and CD8+ T cells following SHIVSF162P3-infection of naïve macaques. Transient co-transfection studies in 293 cells with HXB2 and Nedd4 demonstrated a Nedd4-mediated increase in p24 and gp120 levels. This increase was found to be dependent on the Ca2+/calmodulin-regulated phospholipid binding C2 domain and not ubiquitin ligase activity or HIV LTR activity. Co-transfection of Nedd4 with plasmid DNA expressing Gag or Env was further shown to augment both intracellular and extracellular Gag or Env proteins. To assess the potential of Nedd4 as an immune modulator, BALB/c mice were immunized intramuscularly with plasmid DNA encoding HIV gag, env and Nedd4. Nedd4 co-administration was found to increase serum anti-p24 but not anti-gp120 antibodies. Nedd4 co-injection was found to have no affect on Gag- or Env-specific IFNγ but had a trend of increased Gag-specific IL-6, IL-17A and TNFα that was not seen following Env stimulation. Based on our initial findings, Nedd4-mediated changes in HIV protein levels and its potential use in HIV-1 vaccine development warrants further investigation.

Allosteric induction of the CD4-bound conformation of HIV-1 Gp120

BackgroundHIV-1 infection of target cells is mediated via the binding of the viral envelope protein, gp120, to the cell surface receptor CD4. This interaction leads to conformational rearrangements in gp120 forming or revealing CD4 induced (CD4i) epitopes which are critical for the subsequent recognition of the co-receptor required for viral entry. The CD4-bound state of gp120 has been considered a potential immunogen for HIV-1 vaccine development. Here we report on an alternative means to induce gp120 into the CD4i conformation.ResultsCombinatorial phage display peptide libraries were screened against HIV-1 gp120 and short (14aa) peptides were selected that bind the viral envelope and allosterically induce the CD4i conformation. The lead peptide was subsequently systematically optimized for higher affinity as well as more efficient inductive activity. The peptide:gp120 complex was scrutinized with a panel of neutralizing anti-gp120 monoclonal antibodies and CD4 itself, illustrating that peptide binding does not interfere with or obscure the CD4 binding site.ConclusionsTwo surfaces of gp120 are considered targets for the development of cross neutralizing antibodies against HIV-1; the CD4 binding site and CD4i epitopes. By implementing novel peptides that allosterically induce the CD4i epitopes we have generated a viral envelope that presents both of these surfaces simultaneously.

A CD4 mimic as an HIV entry inhibitor: Pharmacokinetics

To date, several small molecules of CD4 mimics, which can suppress competitively the interaction between an HIV-1 envelope glycoprotein gp120 and a cellular surface protein CD4, have been reported as viral entry inhibitors. A lead compound 2 (YYA-021) with relatively high potency and low cytotoxicity has been identified previously by SAR studies. In the present study, the pharmacokinetics of the intravenous administration of compound 2 in rats and rhesus macaques is reported. The half-lives of compound 2 in blood in rats and rhesus macaques suggest that compound 2 shows wide tissue distribution and relatively high distribution volumes. A few hours after the injection, both plasma concentrations of compound 2 maintained micromolar levels, indicating it might have promise for intravenous administration when used combinatorially with anti-gp120 monoclonal antibodies.

A Cocktail of Thermally Stable, Chemically Synthesized Capture Agents for the Efficient Detection of Anti-Gp41 Antibodies from Human Sera

We report on a method to improve in vitro diagnostic assays that detect immune response, with specific application to HIV-1. The inherent polyclonal diversity of the humoral immune response was addressed by using sequential in situ click chemistry to develop a cocktail of peptide-based capture agents, the components of which were raised against different, representative anti-HIV antibodies that bind to a conserved epitope of the HIV-1 envelope protein gp41. The cocktail was used to detect anti-HIV-1 antibodies from a panel of sera collected from HIV-positive patients, with improved signal-to-noise ratio relative to the gold standard commercial recombinant protein antigen. The capture agents were stable when stored as a powder for two months at temperatures close to 60oC.