Non-neutralizing mAb Research Articles

Comparison of an improved competitive enzyme-linked immunosorbent assay with the World Organization for Animal Health–prescribed serum neutralization assay for detection of antibody to Equine arteritis virus

Equine arteritis virus (EAV) causes contagious equine viral arteritis, characterized by fever, anorexia, conjunctivitis, nasal discharge, dependent edema, abortion, infrequent death in foals, and establishment of the carrier state in stallions. The World Organization for Animal Health (OIE) defines a horse as seropositive if the serum neutralization (SN) antibody titer is ≥1:4 to EAV. However, determining the SN titer is time-consuming and requires specific laboratory facilities, equipment, and technical expertise to perform. Furthermore, interpretation of the SN titer of some sera can be difficult because of nonspecific cellular cytotoxicity of particular samples. Finally, the problem of interlaboratory variation also exists with SN assays. For these reasons, an alternative serologic test is desirable; however, none of the reported tests have equivalent sensitivity and specificity to the SN to be generally adopted. In an attempt to improve on a previously developed competitive enzyme-linked immunosorbent assay (cELISA) using EAV gp5-specific neutralizing monoclonal antibody (mAb) 4B2, the current study developed a modified protocol substituting the non-neutralizing mAb 17B7 for the neutralizing mAb 4B2; this along with several modifications of the test procedure improved the performance of the test. The relative specificity of the revamped cELISA was 99.8% when evaluated with 2,223 SN-negative sera. The relative sensitivity was 95.5% when evaluated with 246 SN-positive sera. This new cELISA was not affected by the presence of non-EAV-specific cytotoxicity in sera as observed in the SN assay. The results indicate that this new cELISA may be a viable alternative to the SN assay and merit additional validation.

Rotavirus Infection of Cells in Culture Induces Activation of RhoA and Changes in the Actin and Tubulin Cytoskeleton

Rotavirus infection induces an increase in [Ca2+]cyto, which in turn may affect the distribution of the cytoskeleton proteins in the infected cell. Changes in microfilaments, including the formation of stress fibers, were observed starting at 0.5 h.p.i. using fluorescent phalloidin. Western blot analysis indicated that RhoA is activated between 0.5 and 1 h.p.i. Neither the phosphorylation of RhoA nor the formation of stress fibers were observed in cells infected with virions pre-treated with an anti-VP5* non-neutralizing mAb, suggesting that RhoA activation is stimulated by the interaction of the virus with integrins forming the cell receptor complex. In addition, the structure of the tubulin cytoskeleton was also studied. Alterations of the microtubules were evident starting at 3 h.p.i. and by 7 h.p.i. when microtubules were markedly displaced toward the periphery of the cell cytoplasm. Loading of rotavirus-infected cells with either a Ca2+ chelator (BAPTA) or transfection with siRNAs to silence NSP4, reversed the changes observed in both the microfilaments and microtubules distribution, but not the appearance of stress fibers. These results indicate that alterations in the distribution of actin microfilaments are initiated early during infection by the activation of RhoA, and that latter changes in the Ca2+ homeostasis promoted by NSP4 during infection may be responsible for other alterations in the actin and tubulin cytoskeleton.

Sub-Domains of Ricin’s B Subunit as Targets of Toxin Neutralizing and Non-Neutralizing Monoclonal Antibodies

The B subunit (RTB) of ricin toxin is a galactose (Gal)−/N-acetylgalactosamine (GalNac)-specific lectin that mediates attachment, entry, and intracellular trafficking of ricin in host cells. Structurally, RTB consists of two globular domains with identical folding topologies. Domains 1 and 2 are each comprised of three homologous sub-domains (α, β, γ) that likely arose by gene duplication from a primordial carbohydrate recognition domain (CRD), although only sub-domains 1α and 2γ retain functional lectin activity. As part of our ongoing effort to generate a comprehensive B cell epitope map of ricin, we report the characterization of three new RTB-specific monoclonal antibodies (mAbs). All three mAbs, JB4, B/J F9 and C/M A2, were initially identified based on their abilities to neutralize ricin in a Vero cell cytotoxicty assay and to partially (or completely) block ricin attachment to cell surfaces. However, only JB4 proved capable of neutralizing ricin in a macrophage apoptosis assay and in imparting passive immunity to mice in a model of systemic intoxication. Using a combination of techniques, including competitive ELISAs, pepscan analysis, differential reactivity by Western blot, as well as affinity enrichment of phage displayed peptides, we tentatively localized the epitopes recognized by the non-neutralizing mAbs B/J F9 and C/M A2 to sub-domains 2α and 2β, respectively. Furthermore, we propose that the epitope recognized by JB4 is within sub-domain 2γ, adjacent to RTB’s high affinity Gal/GalNAc CRD. These data suggest that recognition of RTB’s sub-domains 1α and 2γ are critical determinants of antibody neutralizing activity and protective immunity to ricin.

Different Pattern of Immunoglobulin Gene Usage by HIV-1 Compared to Non-HIV-1 Antibodies Derived from the Same Infected Subject

A biased usage of immunoglobulin (Ig) genes is observed in human anti-HIV-1 monoclonal antibodies (mAbs) resulting probably from compensation to reduced usage of the VH3 family genes, while the other alternative suggests that this bias usage is due to antigen requirements. If the antigen structure is responsible for the preferential usage of particular Ig genes, it may have certain implications for HIV vaccine development by the targeting of particular Ig gene-encoded B cell receptors to induce neutralizing anti-HIV-1 antibodies. To address this issue, we have produced HIV-1 specific and non-HIV-1 mAbs from an infected individual and analyzed the Ig gene usage. Green-fluorescence labeled virus-like particles (VLP) expressing HIV-1 envelope (Env) proteins of JRFL and BaL and control VLPs (without Env) were used to select single B cells for the production of 68 recombinant mAbs. Ten of these mAbs were HIV-1 Env specific with neutralizing activity against V3 and the CD4 binding site, as well as non-neutralizing mAbs to gp41. The remaining 58 mAbs were non-HIV-1 Env mAbs with undefined specificities. Analysis revealed that biased usage of Ig genes was restricted only to anti-HIV-1 but not to non-HIV-1 mAbs. The VH1 family genes were dominantly used, followed by VH3, VH4, and VH5 among anti-HIV-1 mAbs, while non-HIV-1 specific mAbs preferentially used VH3 family genes, followed by VH4, VH1 and VH5 families in a pattern identical to Abs derived from healthy individuals. This observation suggests that the biased usage of Ig genes by anti-HIV-1 mAbs is driven by structural requirements of the virus antigens rather than by compensation to any depletion of VH3 B cells due to autoreactive mechanisms, according to the gp120 superantigen hypothesis.

An engineered mutant of HIV-1 gp120 formulated with adjuvant Quil A promotes elicitation of antibody responses overlapping the CD4-binding site.

A major priority in HIV vaccine research is the development of an immunogen to elicit broadly neutralizing antibodies (NAbs). Monoclonal antibody (mAb) b12 is one of now several broadly neutralizing mAbs that bind epitopes overlapping the CD4-binding site (CD4bs) on HIV-1 gp120 and that serve as templates to engineer effective immunogens. We are exploring a strategy whereby extra glycans are incorporated onto gp120 to occlude the epitopes of non-neutralizing mAbs while maintaining exposure of the b12 site. Immunizing with these so-called hyperglycosylated gp120s is hypothesized to preferentially elicit b12-like NAbs. Here, the effects of two adjuvants, monophosphoryl lipid A (MPL) and Quil A, on eliciting b12-like responses when formulated with a new hyperglycosylated mutant, ΔN2mCHO(Q105N), is presented. Sera from ΔN2mCHO(Q105N)_MPL immunized animals bound the homologous antigen ΔN2mCHO(Q105N) with greater preference than sera from ΔN2mCHO(Q105N)_QuilA immunized animals, demonstrating the modulation of antibody fine specificity by these two adjuvants. We also found that sera from ΔN2mCHO(Q105N)_QuilA immunized animals bound best to a resurfaced HIV gp120 core protein on which non-CD4bs epitopes are substituted with non-HIV residues, suggesting that these sera contain a relatively larger fraction of CD4bs-specific antibodies. Consistent with these data, inhibition assays revealed epitope overlap with the binding sites of the CD4bs-specific antibodies b12, b13 and VRC03. Unexpectedly, these sera did not exhibit significant neutralizing activity against a set of HIV-1 primary strains. Our results show that although formulating mutant ΔN2mCHO(Q105N) with Quil A promotes the elicitation of CD4bs-directed antibodies relative to wild-type gp120, tweaking of the immunization regimen is needed to yield robust, CD4bs-focused NAbs.

Antibody-Induced Conformational Changes in Herpes Simplex Virus Glycoprotein gD Reveal New Targets for Virus Neutralization

As the receptor-binding protein of herpes simplex virus (HSV), gD plays an essential role in virus entry. In its native state, the last 56 amino acids of the ectodomain C terminus (C-term) occlude binding to its receptors, herpesvirus entry mediator (HVEM) and nectin-1. Although it is clear that movement of the C-term must occur to permit receptor binding, we believe that this conformational change is also a key event for triggering later steps leading to fusion. Specifically, gD mutants containing disulfide bonds that constrain the C-term are deficient in their ability to trigger fusion following receptor binding. In this report, we show that two newly made monoclonal antibodies (MAbs), MC2 and MC5, have virus-neutralizing activity but do not block binding of gD to either receptor. In contrast, all previously characterized neutralizing anti-gD MAbs block binding of gD to a receptor(s). Interestingly, instead of blocking receptor binding, MC2 significantly enhances the affinity of gD for both receptors. Several nonneutralizing MAbs (MC4, MC10, and MC14) also enhanced gD-receptor binding. While MC2 and MC5 recognized different epitopes on the core of gD, these nonneutralizing MAbs recognized the gD C-term. Both the neutralizing capacity and rate of neutralization of virus by MC2 are uniquely enhanced when MC2 is combined with MAb MC4, MC10, or MC14. We suggest that MC2 and MC5 prevent gD from performing a function that triggers later steps leading to fusion and that the epitope for MC2 is normally occluded by the C-term of the gD ectodomain.

Fine epitope mapping of monoclonal antibodies against hemagglutinin of a highly pathogenic H5N1 influenza virus using yeast surface display

Highly pathogenic H5N1 avian influenza viruses pose a debilitating pandemic threat. Thus, understanding mechanisms of antibody-mediated viral inhibition and neutralization escape is critical. Here, a robust yeast display system for fine epitope mapping of viral surface hemagglutinin (HA)-specific antibodies is demonstrated. The full-length H5 subtype HA (HA0) was expressed on the yeast surface in a correctly folded conformation, determined by binding of a panel of extensively characterized neutralizing human monoclonal antibodies (mAbs). These mAbs target conformationally-dependent epitopes of influenza A HA, which are highly conserved across H5 clades and group 1 serotypes. By separately displaying HA1 and HA2 subunits on yeast, domain mapping of two anti-H5 mAbs, NR2728 and H5-2A, localized their epitopes to HA1. These anti-H5 mAb epitopes were further fine mapped by using a library of yeast-displayed HA1 mutants and selecting for loss of binding without prior knowledge of potential contact residues. By overlaying key mutant residues that impacted binding onto a crystal structure of HA, the NR2728 mAb was found to interact with a fully surface-exposed contiguous patch of residues at the receptor binding site (RBS), giving insight into the mechanism underlying its potent inhibition of virus binding. The non-neutralizing H5-2A mAb was similarly mapped to a highly conserved H5 strain-specific but poorly accessible location on a loop at the trimer HA interface. These data further augment our toolchest for studying HA antigenicity, epitope diversity and accessibility in response to natural and experimental influenza infection and vaccines.

The B-subunit of ricin toxin is involved in eliciting protective antibodies (106.18)

Abstract The ricin toxin B subunit (RTB) is a galactose-specific lectin that promotes attachment and entry of the ricin enzymatic subunit (RTA) into host cells. RTB is an appealing alternative to RTA-based vaccines, because it is non-toxic and inherently antigenic. However, the degree to which RTB elicits protective immunity against ricin remains unresolved. Here we report that parenteral immunization of mice with RTB elicited high ricin-specific serum antibody titers, yet conferred only partial protection against systemic ricin challenge. Analysis of ~2000 RTB-specific B cell hybridomas identified only a handful of neutralizing monoclonal antibodies (mAbs). Two neutralizing mAbs, 24B11 and SylH3, were shown by passive immunization to be sufficient to protect mice against a 5xLD50 dose of ricin. Both 24B11 and SylH3 blocked ricin attachment to terminal galactose residues and prevented toxin binding to primary macrophages, suggesting that they function by steric hindrance and recognize epitopes located on RTB’s galactose binding sub-domains, 1α or 2γ. By contrast, analysis of four non-neutralizing mAbs revealed that they recognized epitopes situated within sub-domains of RTB not involved in galactose recognition (i.e., 1β, 2α). Based on these data, we propose using specific RTB sub-domains as antigens, rather than RTB itself, as a means to more efficiently elicit neutralizing antibodies and protective immunity against ricin.

Neutralizing Monoclonal Antibody to Edema Toxin and Its Effect on Murine Anthrax

Edema factor (EF) is a component of an anthrax toxin that functions as an adenylate cyclase. Numerous monoclonal antibodies (MAbs) have been reported for the other Bacillus anthracis toxin components, but relatively few to EF have been studied. We report the generation of six murine hybridoma lines producing two IgM and four IgG1 MAbs to EF. Of the six MAbs, only one IgM neutralized EF, as assayed by an increase in cyclic AMP (cAMP) production by Chinese hamster ovary (CHO) cells. Analysis of the variable gene elements revealed that the single neutralizing MAb had a different binding site than the others. There was no competition between the neutralizing IgM and the nonneutralizing IgG MAbs indicative of different specificity. MAb-based capture enzyme-linked immunosorbent assay (ELISA) detected EF in liver lysates from mice infected with B. anthracis Sterne 34F2. Administration of the neutralizing IgM MAb to A/JCr mice lethally infected with B. anthracis strain Sterne had no significant effect on median time to death, but mice treated with the MAb were more likely to survive infection. Combining the neutralizing IgM to EF with a subprotective dose of a neutralizing MAb to protective antigen (PA) prolonged mean time to death of infected mice, suggesting that neutralization of EF and PA could produce synergistic beneficial effects. In summary, the results from our study and literature observations suggest that the majority of Abs to EF are nonneutralizing, but the toxin has some epitopes that can be targeted by the humoral response to generate useful Abs that may contribute to defense against anthrax.

Antibody-mediated neutralization of Ebola virus can occur by two distinct mechanisms.

Human Ebola virus causes severe hemorrhagic fever disease with high mortality and there is no vaccine or treatment. Antibodies in survivors occur early, are sustained, and can delay infection when transferred into nonhuman primates. Monoclonal antibodies (mAbs) from survivors exhibit potent neutralizing activity in vitro and are protective in rodents. To better understand targets and mechanisms of neutralization, we investigated a panel of mAbs shown previously to react with the envelope glycoprotein (GP). While one non-neutralizing mAb recognized a GP epitope in the nonessential mucin-like domain, the rest were specific for GP1, were neutralizing, and could be further distinguished by reactivity with secreted GP. We show that survivor antibodies, human KZ52 and monkey JP3K11, were specific for conformation-dependent epitopes comprising residues in GP1 and GP2 and that neutralization occurred by two distinct mechanisms; KZ52 inhibited cathepsin cleavage of GP whereas JP3K11 recognized the cleaved, fusion-active form of GP.

Antibody-mediated synergy and interference in the neutralization of SARS-CoV at an epitope cluster on the spike protein

Incomplete neutralization of virus, especially when it occurs in the presence of excess neutralizing antibody, represents a biological phenomenon that impacts greatly on antibody-mediated immune prophylaxis of viral infection and on successful vaccine design. To understand the mechanism by which a virus escapes from antibody-mediated neutralization, we have investigated the interactions of non-neutralizing and neutralizing antibodies at an epitope cluster on the spike protein of severe acute respiratory syndrome coronavirus (SARS-CoV). The epitope cluster was mapped at the C-terminus of the spike protein; it consists of structurally intertwined epitopes recognized by two neutralizing monoclonal antibodies (mAbs), 341C and 540C, and a non-neutralizing mAb, 240C. While mAb 341C binds to a mostly linear epitope composed of residues 507PAT 509 and V 349, mAb 240C binds to an epitope that partially overlaps the former by at least two residues (P 507 and A 508). The epitope corresponding to mAb 540C is a conformational one, involving residues L 504 and N 505. In neutralization assays, non-neutralizing 240C disrupted virus neutralization by mAb 341C and/or mAb 540C, whereas a combination of mAbs 341C and 540C blocked virus infectivity synergistically. These findings indicate that the epitope cluster on the spike protein may serve as an evolutionarily conserved platform at which a dynamic interplay between neutralizing and non-neutralizing antibodies occurs, thereby determining the outcome of SARS-CoV infection.

Enhanced macrophage tropism of HIV in brain and lymphoid tissues is associated with sensitivity to the broadly neutralizing CD4 binding site antibody b12

Macrophages in the central nervous system (CNS) and other tissues are an important cellular reservoir for human immunodeficiency virus type 1 (HIV) infection, particularly in the later stages of disease. Macrophage-tropic HIV strains have an enhanced capacity to enter cells expressing low levels of CD4 through mechanisms that are not well understood. Here, we use a panel of primary HIV envelopes from brain and lymphoid tissues to examine the relationship between neutralization sensitivity to reagents targeting the CD4 binding site and virus entry into macrophages. Neutralization assays using pseudotyped viruses showed an association between the capacity of HIV to enter macrophages and increased sensitivity to the broadly neutralizing monoclonal antibody (mAb) b12, which recognizes a conserved epitope overlapping the CD4 binding site, but not sensitivity to soluble CD4 (sCD4) or b6, a non-neutralizing CD4 binding site mAb. Furthermore, loss of an N-linked glycosylation site at position 386 in the V4 region of Env enhanced macrophage tropism together with b12 sensitivity, but not neutralization by sCD4, b6, or a broadly neutralizing AIDS patient serum. These findings suggest that exposure of the b12 epitope, rather than exposure of the CD4 binding site per se, enhances HIV macrophage tropism, possibly by exposing a region on the outer domain of gp120 that is initially recognized by CD4. These findings suggest overlap between specific gp120 determinants in or near the b12 epitope and those conferring macrophage tropism.

A sub-population of high proliferative potential-quiescent human mesenchymal stem cells is under the reversible control of interferon α/β

Type I interferon (IFN) is shown to control the reversible quiescence of a primitive human bone marrow mesenchymal stem cell (MSC) subpopulation. A 24 h pre-treatment of Stro1+/GlycoA- or CD45-/GlycoA- subpopulations with a monoclonal antibody (mAb) against the IFNAR1 chain of the human type I IFN receptor (64G12), or with a polyclonal anti-IFNalpha antibody, resulted in a marked increase in the number of very large colonies (CFU-F >3000 cells) obtained in the presence of low, but necessary, concentrations of bFGF. Over a 2-month culture period, this short activation promoted a faster and greater amplification of mesenchymal progenitors for adipocytes and osteoblasts. Activation correlated with inhibition of STAT1 and STAT2 phosphorylation and of STAT1 nuclear translocation. A non-neutralizing anti-IFNAR1 mAb was ineffective. We demonstrate that control and activated MSCs express ST3GAL3, a sialyltransferase necessary to produce the embryonic antigens SSEA-3 and -4. Interestingly, activated MSC progeny expressed SSEA-3 and -4 at a higher level than control cultures, but this was not correlated with a significant expression of other embryonic markers. As MSCs represent an essential tool in tissue regeneration, the use of 64G12, which rapidly recruits a higher number of primitive cells, might increase amplification safety for cell therapy.

A mutation in the envelope protein fusion loop attenuates mouse neuroinvasiveness of the NY99 strain of West Nile virus

Substitutions were engineered individually and in combinations at the fusion loop, receptor-binding domain and a stem-helix structure of the envelope protein of a West Nile virus strain, NY99, and their effects on mouse virulence and presentation of epitopes recognized by monoclonal antibodies (MAbs) were assessed. A single substitution within the fusion loop (L107F) attenuated mouse neuroinvasiveness of NY99. No substitutions attenuated NY99 neurovirulence. The L107F mutation also abolished binding of a non-neutralizing MAb, 3D9, whose epitope had not been previously identified. MAb 3D9 was subsequently shown to be broadly cross-reactive with other flaviviruses, consistent with binding near the highly conserved fusion loop.

Study on the Porcinophilic Foot-and-Mouth Disease Virus I. Production and Characterization of Monoclonal Antibodies against VP1

Monoclonal antibodies (MAbs) reported here were produced against the porcinophilic foot-and-mouth disease virus (FMDV) that caused the devastating swine disease on 1997 in Taiwan. A panel (25) of MAbs were found to react with VP1 of O/Taiwan/97 (O/97) by ELISA with various potencies. The biological identities of these VP1 reacting MAbs, such as neutralization activity, isotype and capability to distinguish between two serotype O FMDVs, O/97 and O/Taiwan/KM1/99 (O/99), were further analyzed. Eleven out of the total eighteen O/97 neutralizing MAbs were able to neutralize heterologous O/99. Eight O/97 neutralizing and five non-neutralizing MAbs could differentiate two serotype O FMDVs by immunofluorescence assay (IFA) implied that these thirteen MAbs recognized O/97 specific epitope(s). Furthermore, reactivities of the VP1 reacting MAbs with a 29 amino acids synthetic peptide (P29) representing the betaG-betaH loop of VP1 were analyzed by ELISA and fourteen were found positive. MAb clone Q10E-3 reacting strongest with VP1 and P29, neutralizing both but not differentiating two serotype O viruses suggested that the antibody binding site might involve the RGD motif and its C terminal conserved region on betaG-betaH loop. MAbs with diverse characters presented in this study were the first raised against porcinophilic FMDV. The complete set of MAbs may be used for further studies of vaccine, diagnostic methods, prophylaxis, etiological and immunological researches on FMDV.

Characterization of neutralizing antibodies to West Nile virus

We produced nine monoclonal antibodies (MAbs) directed against the West Nile virus E glycoprotein using three different immunization strategies: inactivated virus, naked DNA, and recombinant protein. Most of the MAbs bound to conformation dependent epitopes in domain III of the E protein. Four of the MAbs neutralized WNV infection and bound to the same region of domain III with high affinity. The neutralizing MAbs were obtained from mice immunized with inactivated virus alone or in combination with a DNA plasmid. In contrast, MAbs obtained by immunization with a soluble version of the E glycoprotein did not exhibit neutralizing activity. These non-neutralizing antibodies were cross-reactive with several other flaviviruses, including Saint Louis encephalitis, Japanese encephalitis, Yellow Fever and Powassan viruses. Interestingly, some non-neutralizing MAbs bound with high affinity to domains I or III, indicating that both affinity and the precise epitope recognized by an antibody are important determinants of WNV neutralization.

Molecular and Biological Characterization of Human Monoclonal Antibodies Binding to the Spike and Nucleocapsid Proteins of Severe Acute Respiratory Syndrome Coronavirus

Human monoclonal antibodies (MAbs) were selected from semisynthetic antibody phage display libraries by using whole irradiated severe acute respiratory syndrome (SARS) coronavirus (CoV) virions as target. We identified eight human MAbs binding to virus and infected cells, six of which could be mapped to two SARS-CoV structural proteins: the nucleocapsid (N) and spike (S) proteins. Two MAbs reacted with N protein. One of the N protein MAbs recognized a linear epitope conserved between all published human and animal SARS-CoV isolates, and the other bound to a nonlinear N epitope. These two N MAbs did not compete for binding to SARS-CoV. Four MAbs reacted with the S glycoprotein, and three of these MAbs neutralized SARS-CoV in vitro. All three neutralizing anti-S MAbs bound a recombinant S1 fragment comprising residues 318 to 510, a region previously identified as the SARS-CoV S receptor binding domain; the nonneutralizing MAb did not. Two strongly neutralizing anti-S1 MAbs blocked the binding of a recombinant S fragment (residues 1 to 565) to SARS-CoV-susceptible Vero cells completely, whereas a poorly neutralizing S1 MAb blocked binding only partially. The MAb ability to block S1-receptor binding and the level of neutralization of the two strongly neutralizing S1 MAbs correlated with the binding affinity to the S1 domain. Finally, epitope mapping, using recombinant S fragments (residues 318 to 510) containing naturally occurring mutations, revealed the importance of residue N479 for the binding of the most potent neutralizing MAb, CR3014. The complete set of SARS-CoV MAbs described here may be useful for diagnosis, chemoprophylaxis, and therapy of SARS-CoV infection and disease.

Cell fusion activities of Hantaan virus envelope glycoproteins.

Hantaan virus (HTNV)-infected Vero E6 cells undergo cell fusion with both infected and uninfected cells under low-pH conditions. Flow cytometry and fluorescence microscopy of HTNV-infected Vero E6 cells showed that envelope glycoproteins (GPs) were located both on the cell surface and in the cytoplasm. Neutralizing monoclonal antibodies (MAbs) against the G1 and G2 envelope GPs inhibited cell fusion, whereas nonneutralizing MAbs against G1 or G2 and MAbs against the nucleocapsid protein (NP) did not. Transfected Vero E6 cells that expressed GPs but not those that expressed NP fused and formed syncytia. These results indicate that HTNV GPs act as fusogens at the cell surface. No fusion activity was observed either in infected Vero cells that were passaged more than 150 times or in BHK-21 cells, although GPs appeared to localize to the cell surface. This variability in fusion induction suggests the involvement of host cell factors in the process of cell membrane fusion.

540. Retroviral Mediated Gene Transfer of Hexb in Sandhoff Disease

Top of pageAbstract Vaccines that stimulate broadly neutralizing antibodies to HIV-1 will have a major impact on reducing transmission. However this remains an elusive goal because the antibodies induced by the envelope glycoprotein (Env) do not effectively neutralize primary isolates or viral strains that are important in transmission. Using DNA shuffling and screening technologies, we had identified novel gp120 variants with preferential reactivity to the neutralizing human mAb b12 and significantly weakened binding to non-neutralizing CD4BS-binding mAbs b3 and b6. This phenotype mimics that of the native trimeric envelope complex. Biacore studies of mAb-gp120 interaction demonstrated that the enhanced affinity of b12 in the shuffled variants resulted largely from a decrease in the dissociation rate. DNA shuffling of the 'core' form of gp120, which lacks V1/V2, V3 and parts of C1 and C5, also generated gp120 variants that possessed these antigenic properties. We immunized rabbits with >100 independent gp120 variants using electroporation-mediated DNA vaccination, followed by boosting with a heterologous or homologous recombinant protein antigen. A single-cycle pseudovirus-based assay was used to evaluate the HIV-1 neutralization activities of purified immunoglobulins. About 60% of the full-length gp120 variants induced neutralizing antibodies to the TCLA strain SF162, indicating that the DNA shuffling and screening approach provides novel variants that have gp120-like immunogenicity. Several of the shuffled variants induced antibodies to primary viral isolates and five clones were able to neutralize eight primary isolates in addition to TCLA strains. These shuffled variants are candidates for additional shuffling to screen for further improvements in immunogenicity and for testing using additional vaccine formats, including optimized immunization protocols. DNA shuffling extensively recombines mixtures of parental genes such as the Env coding sequences to create highly functional libraries of shuffled chimeras. We have successfully applied this technology to evolve novel viral phenotypes, to improve the antigenicity of the Hepatitis B virus envelope protein and to generate tetravalent Dengue virus antigens. The application of DNA shuffling to HIV vaccine development provides a new approach for generating novel Env structures as vaccine candidates.

Distinct mechanisms of neutralization by monoclonal antibodies specific for sites in the N-terminal or C-terminal domain of murine leukemia virus SU.

The epitope specificities and functional activities of monoclonal antibodies (MAbs) specific for the murine leukemia virus (MuLV) SU envelope protein subunit were determined. Neutralizing antibodies were directed towards two distinct sites in MuLV SU: one overlapping the major receptor-binding pocket in the N-terminal domain and the other involving a region that includes the most C-terminal disulfide-bonded loop. Two other groups of MAbs, reactive with distinct sites in the N-terminal domain or in the proline-rich region (PRR), did not neutralize MuLV infectivity. Only the neutralizing MAbs specific for the receptor-binding pocket were able to block binding of purified SU and MuLV virions to cells expressing the ecotropic MuLV receptor, mCAT-1. Whereas the neutralizing MAbs specific for the C-terminal domain did not interfere with the SU-mCAT-1 interaction, they efficiently inhibited cell-to-cell fusion mediated by MuLV Env, indicating that they interfered with a postattachment event necessary for fusion. The C-terminal domain MAbs displayed the highest neutralization titers and binding activities. However, the nonneutralizing PRR-specific MAbs bound to intact virions with affinities similar to those of the neutralizing receptor-binding pocket-specific MAbs, indicating that epitope exposure, while necessary, is not sufficient for viral neutralization by MAbs. These results identify two separate neutralization domains in MuLV SU and suggest a role for the C-terminal domain in a postattachment step necessary for viral fusion.