A continuous sequence of more than 70 amino acids is essential for antibody binding to the dominant antigenic site of glycoprotein gp58 of human cytomegalovirus.

Antigenic domain 1 (AD-1) on glycoprotein gp58 of human cytomegalovirus was characterized in detail, using mouse and human monoclonal antibodies as well as human convalescent sera. Series of procaryotically expressed fusion proteins and synthetic peptides of various lengths were used as sources of antigen. Binding of antibodies was found to depend on a continuous sequence of more than 70 amino acids between residues 552 and 635 of gp58. The fine specificities for sequences involved in antibody binding were (i) amino acids 557 to 635 for neutralizing as well as nonneutralizing mouse monoclonal antibodies, (ii) amino acids 552 to 630 for a neutralizing human monoclonal antibody, and (iii) amino acids 557 to 630 for antibodies present in human sera. Experiments involving fragments of AD-1, presented either as procaryotically expressed fusion protein or as synthetic peptides, indicated that the intact structure was required for recognition of AD-1 by antibodies.     

Fine specificity of the human immune response to the major neutralization epitopes expressed on cytomegalovirus gp58/116 (gB), as determined with human monoclonal antibodies.

The humoral immune response to human cytomegalovirus (CMV) membrane glycoprotein gp58/116 (gB) has been studied by establishing cell lines producing specific human monoclonal antibodies. These cell lines were generated from peripheral blood lymphocytes obtained from a healthy carrier. Hybridomas producing gp58/116-specific antibodies were detected by reactivity to procaryotically expressed proteins containing the major neutralizing epitopes of this glycoprotein complex. One antibody, ITC88, which recognized an epitope located between amino acid residues 67 and 86 of gp116, potently neutralized the virus at 1 to 2 micrograms of immunoglobulin G per ml. Only four of the six human antibodies detecting the major neutralizing domain of gp58 neutralized the virus, and none of them required complement for activity. All antibodies that bound mature, processed gp58 recognized a conformational epitope involving sequences between residues 549 and 635. However, small differences existed between the antibodies in the actual minimal requirement for C- and N-terminal parts of this epitope. By peptide mapping with several of the antibodies, the epitope was shown to consist mainly of residues between amino acids 570 to 579 and 606 to 619. Despite the conformational nature of the epitope, the antibodies recognized both reduced and denatured native antigen. Presence of carbohydrates was not required for antigen binding of these gp58-specific human antibodies, but in at least one case, it greatly enhanced antigen recognition, indicating an importance of carbohydrate structures in some epitopes within the major neutralizing specificity of gp58.     

Characterization of a Discontinuous Neutralizing Epitope on Glycoprotein B of Human Cytomegalovirus

Human cytomegalovirus (HCMV) is a ubiquitously distributed pathogen that causes severe disease in immunosuppressed patients and newborn infants infected in utero. The viral envelope glycoprotein B (gB) is an attractive molecule for active vaccination and passive immunoprophylaxis and therapy. Using human monoclonal antibodies (MAbs), we have recently identified antigenic region 4 (AD-4) on gB as an important target for neutralizing antibodies. AD-4 is formed by a discontinuous sequence comprising amino acids 121 to 132 and 344 to 438 of gB of HCMV strain AD169. To map epitopes for human antibodies on this protein domain, we used a three-dimensional (3D) model of HCMV gB to identify surface-exposed amino acids on AD-4 and selected juxtaposed residues for alanine scans. A tyrosine (Y) at position 364 and a lysine (K) at position 379 (the YK epitope), which are immediate neighbors on the AD-4 surface, were found to be essential for binding of the human MAbs. Recognition of AD-4 by sera from HCMV-infected individuals also was largely dependent on these two residues, indicating a general importance for the antibody response against AD-4. A panel of AD-4 recombinant viruses harboring mutations at the crucial antibody binding sites was generated. The viruses showed significantly reduced susceptibility to neutralization by AD-4-specific MAbs or polyclonal AD-4-specific antibodies, indicating that the YK epitope is dominant for the AD-4-specific neutralizing antibody response during infection. To our knowledge, this is the first molecular identification of a functional discontinuous epitope on HCMV gB. Induction of antibodies specific for this epitope may be a desirable goal following vaccination with gB.     

Dextran sulfate blocks antibody binding to the principal neutralizing domain of human immunodeficiency virus type 1 without interfering with gp120-CD4 interactions.

The mechanism of the antiviral activity of sulfated polysaccharides on human immunodeficiency virus type 1 (HIV-1) was investigated by determining the effect of dextran sulfate on the binding of CD4 and several anti-gp120 monoclonal antibodies to both recombinant and cell surface gp120. Dextran sulfate did not interfere with the binding of sCD4 to rgp120 on enzyme-linked immunosorbent assay (ELISA) plates or in solution and did not block sCD4 binding to HIV-1-infected cells expressing gp120 on the cell surface. Dextran sulfate had minimal effects on rgp120 binding to CD4+ cells at concentrations which effectively prevent HIV replication. In contrast, it potently inhibited the binding of both rgp120 and cell surface gp120 to several monoclonal antibodies directed against the principal neutralizing domain of gp120 (V3). In an ELISA format, dextran sulfate enhanced the binding of monoclonal antibodies against amino-terminal regions of gp120 and had no effect on antibodies directed to other regions of gp120, including the carboxy terminus. The inhibitory effects of polyanionic polysaccharides on viral binding, viral replication, and formation of syncytia therefore appear mediated by interactions with positively charged amino acids concentrated in the V3 region. This high local positive charge density, unique to the V3 loop, leads us to propose that this property is critical to the function of the V3 region in mediating envelope binding and subsequent fusion between viral and cell membranes. The specific interaction of dextran sulfate with this domain suggests that structurally related molecules on the cell surface, such as heparan sulfate, may be additional targets for HIV binding and infection.     

Binding of HIV-1 gp41-Directed Neutralizing and Non-Neutralizing Fragment Antibody Binding Domain (Fab) and Single Chain Variable Fragment (ScFv) Antibodies to the Ectodomain of gp41 in the Pre-Hairpin and Six-Helix Bundle Conformations

We previously reported a series of antibodies, in fragment antigen binding domain (Fab) formats, selected from a human non-immune phage library, directed against the internal trimeric coiled-coil of the N-heptad repeat (N-HR) of HIV-1 gp41. Broadly neutralizing antibodies from that series bind to both the fully exposed N-HR trimer, representing the pre-hairpin intermediate state of gp41, and to partially-exposed N-HR helices within the context of the gp41 six-helix bundle. While the affinities of the Fabs for pre-hairpin intermediate mimetics vary by only 2 to 20-fold between neutralizing and non-neutralizing antibodies, differences in inhibition of viral entry exceed three orders of magnitude. Here we compare the binding of neutralizing (8066) and non-neutralizing (8062) antibodies, differing in only four positions within the CDR-H2 binding loop, in Fab and single chain variable fragment (ScFv) formats, to several pre-hairpin intermediate and six-helix bundle constructs of gp41. Residues 56 and 58 of the mini-antibodies are shown to be crucial for neutralization activity. There is a large differential (≥150-fold) in binding affinity between neutralizing and non-neutralizing antibodies to the six-helix bundle of gp41 and binding to the six-helix bundle does not involve displacement of the outer C-terminal helices of the bundle. The binding stoichiometry is one six-helix bundle to one Fab or three ScFvs. We postulate that neutralization by the 8066 antibody is achieved by binding to a continuum of states along the fusion pathway from the pre-hairpin intermediate all the way to the formation of the six-helix bundle, but prior to irreversible fusion between viral and cellular membranes.     

The C108g epitope in the V2 domain of gp120 functions as a potent neutralization target when introduced into envelope proteins derived from human immunodeficiency virus type 1 primary isolates

Monoclonal antibodies (MAbs) directed against epitopes in the V2 domain of human immunodeficiency virus type 1 gp120 often possess neutralizing activity, but these generally are highly type specific, neutralize only laboratory isolates, or have low potency. The most potent of these is C108g, directed against a type-specific epitope in HXB2 and BaL gp120s, which is glycan dependent and, in contrast to previous reports, dependent on intact disulfide bonds. This epitope was introduced into two primary Envs, derived from a neutralization-sensitive (SF162) and a neutralization-resistant (JR-FL) isolate, by substitution of two residues and, for SF162, addition of an N-linked glycosylation site. C108g effectively neutralized both variant Envs with considerably higher potency than standard MAbs against the V3 and CD4-binding domains and the broadly neutralizing MAbs 2G12 and 2F5. These amino acid substitutions also introduced the epitope recognized by a second V2-specific MAb, 10/76b, but this MAb possessed potent neutralizing activity only in the absence of the glycan required for C108g reactivity. In contrast to other gp120-specific neutralizing MAbs, C108g did not block binding of soluble Env proteins to either the CD4 or the CCR5 receptor, but studies with a fusion-arrested Env indicated that C108g neutralized at a step preceding the one blocked by the gp41-specific MAb, 2F5. These results indicate that the V1/V2 domain possesses targets that mediate potent neutralization of primary viral isolates via a novel mechanism and suggest that inclusion of carbohydrate determinants into these epitopes may help overcome the indirect masking effects that limit the neutralizing potency of antibodies commonly produced after infection.     

Trimeric membrane-anchored gp41 inhibits HIV membrane fusion

The HIV-1 envelope glycoprotein is composed of a receptor binding subunit, gp120 that is non-covalently linked to the membrane-anchored fusion protein, gp41. Triggered by cellular receptor binding, the trimeric envelope complex mediates the fusion of viral and cellular membranes through the rearrangement of the fusion protein subunit into a six-helical bundle core structure. Here we describe the biophysical and functional properties of a membrane-anchored fragment of gp41 (gp41ctm) that includes the complete C-terminal heptad repeat region 2, the connecting part, and the transmembrane region. We show that the transmembrane domain of the envelope glycoprotein is sufficient for trimerization in vitro, contributing most of the alpha-helical content of gp41ctm. Trimeric gp41ctm is protease-resistant and recognizes neutralizing antibodies 2F5 and 4E10. However, gp41ctm and gp41ctm proteoliposomes elicit no clear neutralizing immune responses in preliminary mouse studies. We further show that gp41ctm and surprisingly also gp41ctm proteoliposomes have potent anti-viral activity. Our data suggest that liposome-anchored gp41ctm exerts its inhibitory action outside of the initial fusion contact site, and its implications for the fusion reaction are discussed.     

Identification and mapping of the gene encoding the glycoprotein complex gp82-gp105 of human herpesvirus 6 and mapping of the neutralizing epitope recognized by monoclonal antibodies.

Monoclonal antibodies (MAbs) 2D4, 2D6, and 13D6 against human herpesvirus 6 (HHV-6) variant A strain GS recognized virion envelope glycoprotein complex gp82-gp105 and neutralized the infectivity of HHV-6 variant A group isolates. A 624-bp genomic fragment (82G) was identified from an HHV-6 strain GS genomic library constructed in the lambda gt11 expression system by immunoscreening with MAb 2D6. Rabbit antibodies against the fusion protein expressed from the genomic insert recognized glycoprotein complex gp82-gp105 from HHV-6-infected cells, thus confirming that the genomic fragment is a portion of the gene(s) that encodes gp82-gp105. This genomic insert hybridized specifically with viral DNAs from HHV-6 variant A strains GS and U1101 under high-stringency conditions but hybridized with HHV-6 variant B strain Z-29 DNA only under low-stringency conditions. DNA sequence analysis of the insert revealed a 167-amino-acid single open reading frame with an open 5' end and a stop codon at the 3' end. Hybridization studies with HHV-6A strain U1102 DNA localized the gp82-gp105-encoding gene to the unique long region near the direct repeat at the right end of the genome. To locate the neutralizing epitope(s) recognized by the MAbs, a series of deletions from the 3' end of the gene were constructed with exonuclease III, and fusion proteins from deletion constructs were tested for reactivity with MAbs in a Western immunoblot assay. Sequencing of deletion constructs at the reactive-nonreactive transition point localized the epitope recognized by the three neutralizing MAbs within or near a repeat amino acid sequence (NIYFNIY) of the putative protein. This repeat sequence region is surrounded on either side by two potential N-glycosylation sites and three cysteine residues.     

Induction of mucosal and systemic neutralizing antibodies against human immunodeficiency virus type 1 (HIV-1) by oral immunization with bovine Papillomavirus-HIV-1 gp41 chimeric virus-like particles

Human immunodeficiency virus type 1 (HIV-1) envelope-specific neutralizing antibodies are generated late after initial infection, and the neutralizing antibody response is weak in the infected individuals. Administration of neutralizing antibodies such as 2F5 to HIV-1-infected individuals resulted in reductions in viral loads. Because HIV-1 is transmitted mainly via mucosa and because HIV-specific neutralizing antibodies reduce HIV-1 in infected individuals, a vaccine that can induce both mucosal and systemic HIV-1-specific neutralizing antibodies may be used to prevent and to treat HIV-1 infection. In this study, we made a bovine papillomavirus (BPV) L1-HIV-1 gp41 fusion protein in which ELDKWA of gp41 was inserted into the N terminus of BPV L1 (amino acids 130 to 136). Expression of the fusion protein in insect cells led to the assembly of chimeric virus-like particles (CVLPs). The CVLPs had sizes similar to those of BPV particles and were able to bind to the cell surface and penetrate the cell membrane. Oral immunization of mice with CVLPs induced gp41-specific serum immunoglobulin G (IgG) and intestinal secretory IgA. However, intramuscular immunization with the CVLPs resulted in similar amounts of gp41-specific IgG but low levels of secretory IgA. The antibodies specifically recognized the fixed HIV-1 gp41 on the cell surface. Importantly, the sera and fecal extracts from mice orally immunized with the CVLPs neutralized HIV-1(MN) in vitro. Thus, BPV-HIV-1 gp41 CVLPs may be used to prevent and to treat HIV-1 infection.     

Identification and characterization of a neutralization site within the second variable region of human immunodeficiency virus type 1 gp120.

Two monoclonal antibodies designated BAT085 and G3-136 were raised by immunizing BALB/c mice with gp120 purified from human immunodeficiency virus type 1 (HIV-1) IIIB-infected H9 cell extracts. Among three HIV-1 laboratory isolates (IIIB, MN, and RF), BAT085 neutralized only IIIB infection of CEM-SS cells, whereas G3-136 neutralized both IIIB and RF. These antibodies also neutralized a few primary HIV-1 isolates in the infection of activated human peripheral blood mononuclear cells. In indirect immunofluorescence assays, BAT085 bound to H9 cells infected with IIIB or MN, while G3-136 bound to H9 cells infected with IIIB or RF, but not MN. Using sequence-overlapping synthetic peptides of HIV-1 IIIB gp120, the binding site of BAT085 and G3-136 was mapped to a peptidic segment in the V2 region (amino acid residues 169 to 183). The binding of these antibodies to immobilized gp120 was not inhibited by the antibodies directed to the principal neutralization determinant in the V3 region or to the CD4-binding domain of gp120. In a competition enzyme-linked immunosorbent assay, soluble CD4 inhibited G3-136 but not BAT085 from binding to gp120. Deglycosylation of gp120 by endo-beta-N-acetylglucosaminidase H or reduction of gp120 by dithiothreitol diminished its reactivity with G3-136 but not with BAT085. These results indicate that the V2 region of gp120 contains multiple neutralization determinants recognized by antibodies in both a conformation-dependent and -independent manner.     

Human anti-V2 monoclonal antibody that neutralizes primary but not laboratory isolates of human immunodeficiency virus type 1.

A human immunoglobulin G1 lambda monoclonal antibody (MAb), 697-D, was developed that recognizes the V2 region of human immunodeficiency virus type 1 (HIV-1) gp120. Substitutions at amino acid positions 176/177, 179/180, 183/184, and 192 to 194 in the V2 loop of gp120 each completely abolished the binding capacity of 697-D in an enzyme-linked immunosorbent assay format. Competition analysis with three different neutralizing murine anti-V2 MAbs confirmed the specificity of 697-D. The 697-D epitope is primarily conformation dependent, although there was weak reactivity of the MAb with a V2 peptide spanning residues 161 to 180. Treatment of recombinant gp120 HIVIIIB with sodium metaperiodate, which oxidizes carbohydrates, abolished the binding of the MAb, showing the dependence of the epitope on intact carbohydrates. The broad reactivity of 697-D was displayed by its binding to the gp120 molecules from four of four laboratory isolates and five of five primary isolates. The MAb 697-D neutralized three out of four primary isolates but failed to neutralize any of four laboratory strains of HIV-1. 697-D and a human anti-V3 MAb, 447-52-D, displayed similar potency in neutralizing primary isolates, indicating that the V2 region of gp120, like the V3 region and the CD4-binding domain, can induce potent neutralizing antibodies against HIV-1 in humans.     

B cell repertoire analysis identifies new antigenic domains on glycoprotein B of human cytomegalovirus which are target of neutralizing antibodies

Human cytomegalovirus (HCMV), a herpesvirus, is a ubiquitously distributed pathogen that causes severe disease in immunosuppressed patients and infected newborns. Efforts are underway to prepare effective subunit vaccines and therapies including antiviral antibodies. However, current vaccine efforts are hampered by the lack of information on protective immune responses against HCMV. Characterizing the B-cell response in healthy infected individuals could aid in the design of optimal vaccines and therapeutic antibodies. To address this problem, we determined, for the first time, the B-cell repertoire against glycoprotein B (gB) of HCMV in different healthy HCMV seropositive individuals in an unbiased fashion. HCMV gB represents a dominant viral antigenic determinant for induction of neutralizing antibodies during infection and is also a component in several experimental HCMV vaccines currently being tested in humans. Our findings have revealed that the vast majority (>90%) of gB-specific antibodies secreted from B-cell clones do not have virus neutralizing activity. Most neutralizing antibodies were found to bind to epitopes not located within the previously characterized antigenic domains (AD) of gB. To map the target structures of these neutralizing antibodies, we generated a 3D model of HCMV gB and used it to identify surface exposed protein domains. Two protein domains were found to be targeted by the majority of neutralizing antibodies. Domain I, located between amino acids (aa) 133-343 of gB and domain II, a discontinuous domain, built from residues 121-132 and 344-438. Analysis of a larger panel of human sera from HCMV seropositive individuals revealed positivity rates of >50% against domain I and >90% against domain II, respectively. In accordance with previous nomenclature the domains were designated AD-4 (Dom II) and AD-5 (Dom I), respectively. Collectively, these data will contribute to optimal vaccine design and development of antibodies effective in passive immunization.     

Glycoprotein N of Human Cytomegalovirus Protects the Virus from Neutralizing Antibodies

Herpes viruses persist in the infected host and are transmitted between hosts in the presence of a fully functional humoral immune response, suggesting that they can evade neutralization by antiviral antibodies. Human cytomegalovirus (HCMV) encodes a number of polymorphic highly glycosylated virion glycoproteins (g), including the essential envelope glycoprotein, gN. We have tested the hypothesis that glycosylation of gN contributes to resistance of the virus to neutralizing antibodies. Recombinant viruses carrying deletions in serine/threonine rich sequences within the glycosylated surface domain of gN were constructed in the genetic background of HCMV strain AD169. The deletions had no influence on the formation of the gM/gN complex and in vitro replication of the respective viruses compared to the parent virus. The gN-truncated viruses were significantly more susceptible to neutralization by a gN-specific monoclonal antibody and in addition by a number of gB- and gH-specific monoclonal antibodies. Sera from individuals previously infected with HCMV also more efficiently neutralized gN-truncated viruses. Immunization of mice with viruses that expressed the truncated forms of gN resulted in significantly higher serum neutralizing antibody titers against the homologous strain that was accompanied by increased antibody titers against known neutralizing epitopes on gB and gH. Importantly, neutralization activity of sera from animals immunized with gN-truncated virus did not exhibit enhanced neutralizing activity against the parental wild type virus carrying the fully glycosylated wild type gN. Our results indicate that the extensive glycosylation of gN could represent a potentially important mechanism by which HCMV neutralization by a number of different antibody reactivities can be inhibited.     

Variant-specific monoclonal and group-specific polyclonal human immunodeficiency virus type 1 neutralizing antibodies raised with synthetic peptides from the gp120 third variable domain.

The third variable (V3) domain of the human immunodeficiency virus type 1 (HIV-1) external membrane glycoprotein gp120 is of crucial importance in eliciting neutralizing antibodies in infected persons. Polyclonal (PAb) and monoclonal (MAb) antibodies directed against selected epitopes in the V3 domain are valuable tools for analysis of the involvement of such sequences in neutralization and for definition of the relation between amino acid variability and immunological cross-reactions. The aim of this study was to obtain such site-specific antibodies. By using synthetic peptides derived from the V3 domain, a group-specific neutralizing PAb, two high-affinity HIV-1 IIIB neutralizing MAb, and two nonneutralizing MAb were raised. A 15-amino-acid peptide overlapping the tip of the V3 domain of HIV-1 MN was used to produce a rabbit PAb (W0/07). This PAb inhibited syncytium formation induced by HIV-1 IIIB and four field isolates. A similar IIIB-derived peptide was used to generate two murine immunoglobulin G1 (IgG1) MAb (IIIB-V3-13 and IIIB-V3-34). Pepscan analysis mapped the binding site of IIIB-V3-34 to the sequence IRIQRGPGR. The Kds of IIIB-V3-13 and IIIB-V3-34 for gp120 were 6.8 x 10(-11) and 1.6 x 10(-10) M, respectively. These MAb neutralized IIIB but not MN and inhibited syncytium formation induced by IIIB. They are applicable in enzyme-linked immunosorbent assays, immunocytochemistry, and flow cytometry. A peptide covering the left base of the V3 domain was used to generate two murine IgG1 MAb (IIIB-V3-21 and IIIB-V3-26). The binding site of IIIB-V3-21 was mapped to the sequence INCTRPN. These MAb did not neutralize HIV-1 and did not inhibit syncytium formation. This study supports the notion that HIV-1 neutralizing antibodies suitable for multiassay performance can be obtained with synthetic peptides and that high-affinity MAb can be generated. Such site-specific antibodies are useful reagents in the analysis of HIV-1 neutralization. In addition, the cross-neutralization of different viral strains by PAb generated through single-peptide immunization is directly relevant to vaccine development.     

Immunization with a soluble CD4-gp120 complex preferentially induces neutralizing anti-human immunodeficiency virus type 1 antibodies directed to conformation-dependent epitopes of gp120.

Preservation of the conformation of recombinant gp120 in an adjuvant, enabling it to elicit conformation-dependent, epitope-specific, broadly neutralizing antibodies, may be critical for the development of any gp120-based human immunodeficiency virus type 1 (HIV-1) vaccine. It was hypothesized that recombinant gp120 complexed with recombinant CD4 could stabilize the conformation-dependent neutralizing epitopes and effectively deliver them to the immune system. Therefore, a soluble CD4-gp120 complex in Syntex adjuvant formulation was tested with mice for its ability to induce neutralizing anti-gp120 antibody responses. Seventeen monoclonal antibodies (MAbs) were generated and characterized. Immunochemical studies, neutralization assays, and mapping studies with gp120 mutants indicated that the 17 MAbs fell into three groups. Four of them were directed to what is probably a conformational epitope involving the C1 domain and did not possess virus-neutralizing activities. Another four MAbs bound to V3 peptide 302-321 and exhibited cross-reactive gp120 binding and relatively weak virus-neutralizing activities. These MAbs were very sensitive to amino acid substitutions, not only in the V3 regions but also in the base of the V1/V2 loop, implying a conformational constraint on the epitope. The last group of nine MAbs recognized conformation-dependent epitopes near the CD4 binding site of gp120 and inhibited the gp120-soluble CD4 interaction. Four of these nine MAbs showed broadly neutralizing activities against multiple laboratory-adapted strains of HIV-1, three of them neutralized only HIVIIIB, and the two lower-affinity MAbs did not neutralize any strain tested. Collectively, the results from this study indicate that immunization with the CD4-gp120 complex can elicit antibodies to conformationally sensitive gp120 epitopes, with some of the antibodies having broadly neutralizing activities. We suggest that immunization with CD4-gp120 complexes may be worth evaluating further for the development of an AIDS vaccine.     

Trimeric gp120-specific bovine monoclonal antibodies require cysteine and aromatic residues in CDRH3 for high affinity binding to HIV Env

We isolated HIV-1 Envelope (Env)-specific memory B cells from a cow that had developed high titer polyclonal immunoglobulin G (IgG) with broad neutralizing activity after a long duration vaccination with HIV-1_AD8 Env gp140 trimers. We cloned the bovine IgG matched heavy (H) and light (L) chain variable (V) genes from these memory B cells and constructed IgG monoclonal antibodies (mAbs) with either a human constant (C)-region/bovine V-region chimeric or fully bovine C and V regions. Among 42 selected Ig+ memory B cells, two mAbs (6A and 8C) showed high affinity binding to gp140 Env. Characterization of both the fully bovine and human chimeric isoforms of these two mAbs revealed them as highly type-specific and capable of binding only to soluble AD8 uncleaved gp140 trimers and covalently stabilized AD8 SOSIP gp140 cleaved trimers, but not monomeric gp120. Genomic sequence analysis of the V genes showed the third heavy complementarity-determining region (CDRH3) of 6A mAb was 21 amino acids in length while 8C CDRH3 was 14 amino acids long. The entire V heavy (VH) region was 27% and 25% diverged for 6A and 8C, respectively, from the best matched germline V genes available, and the CDRH3 regions of 6A and 8C were 47.62% and 78.57% somatically mutated, respectively, suggesting a high level of somatic hypermutation compared with CDRH3 of other species. Alanine mutagenesis of the VH genes of 6A and 8C, showed that CDRH3 cysteine and tryptophan amino acids were crucial for antigen binding. Therefore, these bovine vaccine-induced anti-HIV antibodies shared some of the notable structural features of elite human broadly neutralizing antibodies, such as CDRH3 size and somatic mutation during affinity-maturation. However, while the 6A and 8C mAbs inhibited soluble CD4 binding to gp140 Env, they did not recapitulate the neutralizing activity of the polyclonal antibodies against HIV infection.     

Point Mutations in the Paramyxovirus F Protein That Enhance Fusion Activity Shift the Mechanism of Complement-Mediated Virus Neutralization

Parainfluenza virus 5 (PIV5) activates and is neutralized by the alternative pathway (AP) in normal human serum (NHS) but not by heat-inactivated (HI) serum. We have tested the relationship between the fusion activity within the PIV5 F protein, the activation of complement pathways, and subsequent complement-mediated virus neutralization. Recombinant PIV5 viruses with enhanced fusion activity were generated by introducing point mutations in the F fusogenic peptide (G3A) or at a distal site near the F transmembrane domain (S443P). In contrast to wild-type (WT) PIV5, the mutant G3A and S443P viruses were neutralized by both NHS and HI serum. Unlike WT PIV5, hyperfusogenic G3A and S443P viruses were potent C4 activators, C4 was deposited on NHS-treated mutant virions, and the mutants were neutralized by factor B-depleted serum but not by C4-depleted serum. Antibodies purified from HI human serum were sufficient to neutralize both G3A and S443P viruses in vitro but were ineffective against WT PIV5. Electron microscopy data showed greater deposition of purified human antibodies on G3A and S443P virions than on WT PIV5 particles. These data indicate that single amino acid changes that enhance the fusion activity of the PIV5 F protein shift the mechanism of complement activation in the context of viral particles or on the surface of virus-infected cells, due to enhanced binding of antibodies. We present general models for the relationship between enhanced fusion activity in the paramyxovirus F protein and increased susceptibility to antibody-mediated neutralization.     

Nonneutralizing Antibodies Induced by the HIV-1 gp41 NHR Domain Gain Neutralizing Activity in the Presence of the HIV Fusion Inhibitor Enfuvirtide: a Potential Therapeutic Vaccine Strategy

A key barrier against developing preventive and therapeutic human immunodeficiency virus (HIV) vaccines is the inability of viral envelope glycoproteins to elicit broad and potent neutralizing antibodies. However, in the presence of fusion inhibitor enfuvirtide, we show that the nonneutralizing antibodies induced by the HIV type 1 (HIV-1) gp41 N-terminal heptad repeat (NHR) domain (N63) exhibit potent and broad neutralizing activity against laboratory-adapted HIV-1 strains, including the drug-resistant variants, and primary HIV-1 isolates with different subtypes, suggesting the potential of developing gp41-targeted HIV therapeutic vaccines.     

Cryo-electron tomographic structure of an immunodeficiency virus envelope complex in situ

The envelope glycoprotein (Env) complexes of the human and simian immunodeficiency viruses (HIV and SIV, respectively) mediate viral entry and are a target for neutralizing antibodies. The receptor binding surfaces of Env are in large part sterically occluded or conformationally masked prior to receptor binding. Knowledge of the unliganded, trimeric Env structure is key for an understanding of viral entry and immune escape, and for the design of vaccines to elicit neutralizing antibodies. We have used cryo-electron tomography and averaging to obtain the structure of the SIV Env complex prior to fusion. Our result reveals novel details of Env organisation, including tight interaction between monomers in the gp41 trimer, associated with a three-lobed, membrane-distal gp120 trimer. A cavity exists at the gp41-gp120 trimer interface. Our model for the spike structure agrees with previously predicted interactions between gp41 monomers, and furthers our understanding of gp120 interactions within an intact spike.     

Mechanistic Study of Broadly Neutralizing Human Monoclonal Antibodies against Dengue Virus That Target the Fusion Loop

There are no available vaccines for dengue, the most important mosquito-transmitted viral disease. Mechanistic studies with anti-dengue virus (DENV) human monoclonal antibodies (hMAbs) provide a rational approach to identify and characterize neutralizing epitopes on DENV structural proteins that can serve to inform vaccine strategies. Here, we report a class of hMAbs that is likely to be an important determinant in the human humoral response to DENV infection. In this study, we identified and characterized three broadly neutralizing anti-DENV hMAbs: 4.8A, D11C, and 1.6D. These antibodies were isolated from three different convalescent patients with distinct histories of DENV infection yet demonstrated remarkable similarities. All three hMAbs recognized the E glycoprotein with high affinity, neutralized all four serotypes of DENV, and mediated antibody-dependent enhancement of infection in Fc receptor-bearing cells at subneutralizing concentrations. The neutralization activities of these hMAbs correlated with a strong inhibition of virus-liposome and intracellular fusion, not virus-cell binding. We mapped epitopes of these antibodies to the highly conserved fusion loop region of E domain II. Mutations at fusion loop residues W101, L107, and/or G109 significantly reduced the binding of the hMAbs to E protein. The results show that hMAbs directed against the highly conserved E protein fusion loop block viral entry downstream of virus-cell binding by inhibiting E protein-mediated fusion. Characterization of hMAbs targeting this region may provide new insights into DENV vaccine and therapeutic strategies.