Characterization of the Borna disease virus phosphoprotein, p23.

Borna disease virus infection is diagnosed by the presence of serum antibodies reactive with the major viral proteins, p40 and p23. Although p40 and p23 are unrelated in amino acid sequence structure, cross-reactive antibodies are described. Protein fragments and synthetic peptides were analyzed to characterize the specificities of antibodies to p23. Epitope mapping revealed eight continuous epitopes accessible on the surface of a predicted structural model for the monomeric and the disulfide-linked dimeric forms of p23. None of these epitopes was reactive with antibodies to p40. Cross-reactivity with monospecific sera and monoclonal antibodies to p40 was found for one discontinuous epitope located at the amino terminus of p23.     

Antibody neutralization escape mediated by point mutations in the intracytoplasmic tail of human immunodeficiency virus type 1 gp41

The persistence of human immunodeficiency virus type 1 (HIV-1) infection in the presence of robust host immunity has been associated in part with variation in viral envelope proteins leading to antigenic variation and escape from neutralizing antibodies. Previous studies of natural neutralization escape mutants have predominantly focused on gp120 and gp41 ectodomain sequence variations that alter antibody binding via changes in conformation or glycosylation pattern of the Env, likely due to the immune pressure exerted on the exposed ectodomain component of the glycoprotein. Here, we show for the first time a novel mechanism by which point mutations in the intracytoplasmic tail of the transmembrane component (gp41) of envelope can render the virus resistant to neutralization by monoclonal antibodies and broadly neutralizing polyclonal serum antibodies. Point mutations in a highly conserved structural motif within the intracytoplasmic tail resulted in decreased binding of neutralizing antibodies to the Env ectodomain, evidently due to allosteric changes both in the gp41 ectodomain and in gp120. While receptor binding and infectivity of the mutant virus remained unaltered, the changes in Env antigenicity were associated with an increase in neutralization resistance of the mutant virus. These studies demonstrate the structurally integrated nature of gp120 and gp41 and underscore a previously unrecognized potentially critical role for even minor sequence variation of the intracytoplasmic tail in modulating the antigenicity of the ectodomain of HIV-1 envelope glycoprotein complex.     

Biochemical and functional analysis of the Borna disease virus G protein.

The Borna disease virus (BDV) antigenome is comprised of five major open reading frames (ORFs). Products have been reported only for ORFs I, II, and III, encoding N (p40), P (p24/p23), and M (gp18), respectively. ORF IV predicts a 57-kDa protein with several potential glycosylation sites. Analysis of radiolabeled extracts from BDV-infected C6 cells and BHK-21 cells transfected with a Semliki Forest virus vector that contains ORF IV demonstrated the presence of a 94-kDa protein (G protein) which was sensitive to tunicamycin, endoglycosidase F/N-glycosidase, and endoglycosidase H but not to O-glycosidase. Sera from BDV-infected rats detected the G protein and had neutralization activity that was reduced following immunoadsorption with the G protein. Preincubation of cells with the G protein interfered with BDV infectivity. This effect was enhanced by treatment of the G protein with the exoglycosidase alpha-mannosidase and reduced after subsequent treatment with N-acetyl-beta-D-glucosaminidase. In concert these findings indicate that ORF IV encodes a 94-kDa N-linked glycoprotein with extensive high mannose- and/or hybrid-type oligosaccharide modifications. The presence of neutralization epitopes on the G protein and its capacity to interfere with infectivity suggest that the G protein is important for viral entry.     

HTLV-III neutralizing antibody development in transfusion-dependent seropositive patients with beta-thalassemia

Sera collected in New York in 1984 from 77 patients with homozygous beta-thalassemia were assayed for antibodies to HTLV-III by ELISA and Western blot techniques. Eight (12%) of the 66 hypertransfused thalassemics were seropositive. Retrospective sera of these eight individuals were examined by radioimmune precipitation (RIP), and assays for neutralization of virus infectivity were performed. With seroconversion, antibodies to viral envelope proteins appeared first and were correlated with development of neutralizing antibody. Affinity purified gp120, the major envelope glycoprotein of HTLV-III, blocked viral infectivity and absorbed neutralizing antibody activity from a positive serum. Neutralizing antibody titers mirrored antibody titers to gp120 by RIP. Antibody to gp120 sometimes occurred in the absence of neutralizing antibody, although the reverse was not true. One thalassemia patient who exhibited antibody to gp120 for 3 yr post-seroconversion failed to develop neutralizing antibody, acquired the acquired immunodeficiency syndrome with central nervous system involvement and lymphoma, and subsequently died. In contrast, all other seropositive thalassemics possessed neutralizing antibodies, and were asymptomatic or exhibited only lymphadenopathy. These results indicate that gp120 elicits neutralizing antibodies in the course of natural infection with HTLV-III. The relationship seen here between neutralizing antibody and better clinical outcome needs to be verified by additional studies.     

Neutralizing antibodies in persistent borna disease virus infection: prophylactic effect of gp94-specific monoclonal antibodies in preventing encephalitis

Borna disease virus (BDV) infection triggers an immune-mediated encephalomyelitis and results in a persistent infection. The immune response in the acute phase of the disease is characterized by a cellular response in which CD8(+) T cells are responsible for the destruction of virus-infected brain cells. CD4(+) T cells function as helper cells and support the production of antiviral antibodies. Antibodies generated in the acute phase of the disease against the nucleoprotein and the phosphoprotein are nonneutralizing. In the chronic phase of the disease, neutralizing antibodies directed against the matrix protein and glycoprotein are synthesized. In the present work, the biological role of the neutralizing-antibody response to BDV was further investigated. By analyzing the blood of rats infected intracerebrally with BDV, a highly neurotropic virus, nucleic acid could be detected between 30 and 50 days after infection. Neutralizing antibodies were found between 60 and 100 days after infection. Furthermore, we produced hybridomas secreting BDV-specific neutralizing monoclonal antibodies. These antibodies, directed against the major glycoprotein (gp94) of BDV, were able to prevent Borna disease if given prophylactically. These data suggest that the late appearance of BDV-specific neutralizing antibodies is due to the presence of BDV in the blood of chronically infected rats. Furthermore, these antibodies have the potential to neutralize the infectious virus when given early, which is an important finding with respect to the development of a vaccine.     

Induction and Characterization of Neutralizing Antibodies against a Human Immunodeficiency Virus Type 1 Primary Isolate

Chimpanzees infected with the primary isolate DH012 mount potent neutralizing antibodies. This DH012 neutralizing activity is highly strain specific. Immune sera from guinea pigs immunized with recombinant DH012 gp120 could also neutralize this primary isolate. The neutralizing activity in chimpanzee and guinea pig sera against wild-type DH012 appears to be independent of a linear epitope in the V3 region of gp120. Interestingly, the neutralization escape mutant derived from growing DH012 in the presence of the potent neutralizing chimpanzee serum is at least 50-fold more sensitive than wild-type DH012 to neutralization by guinea pig immune sera. The unusually potent neutralizing activity against the DH012 neutralization-resistant virus is due to the presence of anti-V3 antibodies in guinea pig sera. These results suggested that recombinant gp120 could induce neutralizing antibodies against primary isolate DH012. The V3 of wild-type DH012 is poorly immunogenic in infected chimpanzees and is not accessible to neutralizing V3 antibodies. It is likely that this cryptic V3 region became exposed when the virus escaped the neutralizing activity of the chimpanzee serum.     

Dissecting the neutralizing antibody specificities of broadly neutralizing sera from human immunodeficiency virus type 1-infected donors

Attempts to elicit broadly neutralizing antibody responses by human immunodeficiency virus type 1 (HIV-1) vaccine antigens have been met with limited success. To better understand the requirements for cross-neutralization of HIV-1, we have characterized the neutralizing antibody specificities present in the sera of three asymptomatic individuals exhibiting broad neutralization. Two individuals were infected with clade B viruses and the third with a clade A virus. The broadly neutralizing activity could be exclusively assigned to the protein A-reactive immunoglobulin G (IgG) fraction of all three donor sera. Neutralization inhibition assays performed with a panel of linear peptides corresponding to the third hypervariable (V3) loop of gp120 failed to inhibit serum neutralization of a panel of HIV-1 viruses. The sera also failed to neutralize chimeric simian immunodeficiency virus (SIV) and HIV-2 viruses displaying highly conserved gp41-neutralizing epitopes, suggesting that antibodies directed against these epitopes likely do not account for the broad neutralizing activity observed. Polyclonal IgG was fractionated on recombinant monomeric clade B gp120, and the neutralization capacities of the gp120-depleted samples were compared to that of the original polyclonal IgG. We found that the gp120-binding antibody population mediated neutralization of some isolates, but not all. Overall, the data suggest that broad neutralization results from more than one specificity in the sera but that the number of these specificities is likely small. The most likely epitope recognized by the monomeric gp120 binding neutralizing fraction is the CD4 binding site, although other epitopes, such as the glycan shield, cannot be excluded.     

Human immunodeficiency virus neutralizing antibodies recognize several conserved domains on the envelope glycoproteins.

Serum neutralizing antibodies against the human immunodeficiency virus were frequently detected in infected individuals, and low or absent serum neutralizing titers correlated with poor prognosis. Multiple diverse human immunodeficiency virus isolates were found to exhibit similar susceptibility to neutralization by a panel of human seropositive sera, suggesting that neutralizing antibodies are largely directed against conserved viral domains. Furthermore, utilizing antisera raised against a library of synthetic env peptides, four regions which are important in the neutralization process have been identified within both human immunodeficiency virus envelope glycoproteins (gp41 and gp120). Three of these are in conserved domains and should be considered for inclusion in a candidate vaccine.     

Characterization of antibody responses elicited by human immunodeficiency virus type 1 primary isolate trimeric and monomeric envelope glycoproteins in selected adjuvants

We previously reported that soluble, stable YU2 gp140 trimeric human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein immunogens could elicit improved breadth of neutralization against HIV-1 isolates compared to monomeric YU2 gp120 proteins. In this guinea pig immunization study, we sought to extend these data and determine if adjuvant could quantitatively or qualitatively alter the neutralizing response elicited by trimeric or monomeric immunogens. Consistent with our earlier studies, the YU2 gp140 immunogens elicited higher-titer neutralizing antibodies against homologous and heterologous isolates than those elicited by monomeric YU2 gp120. Additionally, the GlaxoSmithKline family of adjuvants AS01B, AS02A, and AS03 induced higher levels of neutralizing antibodies compared to emulsification of the same immunogens in Ribi adjuvant. Further analysis of vaccine sera indicated that homologous virus neutralization was not mediated by antibodies to the V3 loop, although V3 loop-directed neutralization could be detected for some heterologous isolates. In most gp120-inoculated animals, the homologous YU2 neutralization activity was inhibited by a peptide derived from the YU2 V1 loop, whereas the neutralizing activity elicited by YU2 gp140 trimers was much less sensitive to V1 peptide inhibition. Consistent with a less V1-focused antibody response, sera from the gp140-immunized animals more efficiently neutralized heterologous HIV-1 isolates, as determined by two distinct neutralization formats. Thus, there appear to be qualitative differences in the neutralizing antibody response elicited by YU2 gp140 compared to YU2 monomeric gp120. Further mapping analysis of more conserved regions of gp120/gp41 may be required to determine the neutralizing specificity elicited by the trimeric immunogens.     

Neutralizing Capacity of Monoclonal Antibodies That Recognize Peptide Sequences Underlying the Carbohydrates on gp41 of Simian Immunodeficiency Virus

Extensive glycosylation of the envelope spikes of human and simian immunodeficiency virus (HIV and SIV) is an important factor for the resistance of these viruses to neutralization by antibodies. SIVmac239 gp41 has three closely spaced sites for N-linked carbohydrate attachment. Rhesus macaques experimentally infected with mutant versions of SIVmac239 lacking two or three of these carbohydrate sites developed strong serum reactivity against mutated peptide sequences at the site of these glycosylations, as well as high titers of neutralizing activity to the mutant viruses (E. Yuste et al., J. Virol. 82:12472–12486, 2008). However, whether antibodies that recognize these underlying peptides have neutralizing activity has not been directly demonstrated. Here we describe the isolation and characterization of three gp41-specific monoclonal antibodies (4G8, 6G8, and 7D6) from one of these mutant-infected monkeys. All three antibodies reacted with mutant gp41 from viral particles and also with peptides corresponding to mutated sequences. Slight differences in peptide specificities were observed among the three antibodies. Sequence analysis revealed that the heavy chains of all three antibodies were derived from the same germ line heavy-chain segment (IGHV4-59*01), but they all had very different sequences in complementarity-determining region 3. The light chains of all three antibodies were very closely related to one another. All three antibodies had neutralizing activity to mutant viruses deficient in gp41 carbohydrate attachment, but they did not neutralize the parental SIVmac239. These results demonstrate unambiguously that antibodies with specificity for peptide sequences underlying gp41 carbohydrates can effectively neutralize SIV when these carbohydrates are absent. However, the presence of these gp41 carbohydrates effectively shields the virus from antibodies that would otherwise neutralize viral infectivity.     

Structure and design of broadly-neutralizing antibodies against HIV

Since the discovery more than 30 years ago of human immunodeficiency virus (HIV) as the causative agent of the deadly disease, acquired immune deficiency disease (AIDS), there have been no efficient vaccines against the virus. For the infection of the virus, the HIV surface glycoprotein gp120 first recognizes the CD4 receptor on the target helper T-cell, which initiates HIV fusion with the target cell and, if unchecked, leads to destruction of the patient's immune system. Despite the difficulty of developing appropriate immune responses in HIV-infected individuals, patient sera often contain antibodies that have broad neutralization activity, indicating the possibility of immunological treatment and prevention. Recently, through extensive structural studies of neutralizing antibodies of HIV in complex with gp120, the critical mechanisms of broad neutralization against HIV have been elucidated. Based on these discoveries, the structure-aided designs of antibodies and novel scaffolds were performed to create extremely potent neutralizing antibodies against HIV. These new discoveries and advances shed light on the road to development of efficient immunological therapies against AIDS.     

Nature of nonfunctional envelope proteins on the surface of human immunodeficiency virus type 1

Human immunodeficiency virus type 1 (HIV-1) neutralizing antibodies are thought be distinguished from nonneutralizing antibodies by their ability to recognize functional gp120/gp41 envelope glycoprotein (Env) trimers. The antibody responses induced by natural HIV-1 infection or by vaccine candidates tested to date consist largely of nonneutralizing antibodies. One might have expected a more vigorous neutralizing response, particularly against virus particles that bear functional trimers. The recent surprising observation that nonneutralizing antibodies can specifically capture HIV-1 may provide a clue relating to this paradox. Specifically, it was suggested that forms of Env, to which nonneutralizing antibodies can bind, exist on virus surfaces. Here, we present evidence that HIV-1 particles bear nonfunctional gp120/gp41 monomers and gp120-depleted gp41 stumps. Using a native electrophoresis band shift assay, we show that antibody-trimer binding predicts neutralization and that the nonfunctional forms of Env may account for virus capture by nonneutralizing antibodies. We hypothesize that these nonfunctional forms of Env on particle surfaces serve to divert the antibody response, helping the virus to evade neutralization.     

A cell surface protein that binds avian hepatitis B virus particles.

We have identified a 180-kDa cellular glycoprotein (gp180) that binds with high affinity to duck hepatitis B virus (DHBV) particles. The protein was detected by coprecipitating labeled duck hepatocyte proteins with virions or recombinant DHBV envelope proteins, using nonneutralizing monoclonal antibodies to the virion envelope. Binding of gp180 requires only the pre-S region of the viral large envelope protein, since recombinant fusion proteins bearing only this region efficiently coprecipitate gp180. The DHBV-gp180 interaction is blocked by two independent neutralizing monoclonal antibodies. The protein is found on both internal and surface membranes of the cell, and the species distribution of gp180 binding activity mirrors the known host range of DHBV infection. Functional gp180 is expressed in a wide variety of tissues in susceptible ducks.     

Rational design of a CD4 mimic that inhibits HIV-1 entry and exposes cryptic neutralization epitopes

The conserved surfaces of the human immunodeficiency virus (HIV)-1 envelope involved in receptor binding represent potential targets for the development of entry inhibitors and neutralizing antibodies. Using structural information on a CD4-gp120-17b antibody complex, we have designed a 27-amino acid CD4 mimic, CD4M33, that presents optimal interactions with gp120 and binds to viral particles and diverse HIV-1 envelopes with CD4-like affinity. This mini-CD4 inhibits infection of both immortalized and primary cells by HIV-1, including primary patient isolates that are generally resistant to inhibition by soluble CD4. Furthermore, CD4M33 possesses functional properties of CD4, including the ability to unmask conserved neutralization epitopes of gp120 that are cryptic on the unbound glycoprotein. CD4M33 is a prototype of inhibitors of HIV-1 entry and, in complex with envelope proteins, a potential component of vaccine formulations, or a molecular target in phage display technology to develop broad-spectrum neutralizing antibodies.     

Identification with monoclonal antibodies of glycoproteins of Marek''s disease virus and herpesvirus of turkeys related to virus neutralization.

By use of monoclonal antibodies cross-reactive with Marek's disease virus and herpesvirus of turkeys, three glycoproteins (for Marek's disease virus, gp115/110, gp63, and gp50; for herpesvirus of turkeys, gp115, gp62 and gp52) related to virus neutralization were identified. Immunization of chickens or rabbits with these glycoproteins purified by affinity chromatography resulted in production of neutralizing antibodies.     

Quantitation of HLA proteins incorporated by human immunodeficiency virus type 1 and assessment of neutralizing activity of anti-HLA antibodies

Human anti-human leukocyte antigen (HLA) antibodies were assessed for neutralizing activity against human immunodeficiency virus type 1 (HIV-1) carrying HLA alleles with matching specificity. Multiparous women carrying anti-HLA antibodies were identified. Plasma samples from those women were confirmed as having antibodies that specifically bound to HLA proteins expressed on the peripheral blood mononuclear cells (PBMCs) of their husbands. A primary HIV-1 isolate was cultured in the husband's PBMCs so that the virus carried matching HLA alleles. To determine the HIV-1-neutralizing activity of anti-HLA antibodies, the infectivity of the virus for GHOST cells (which express green fluorescent protein after HIV infection) was investigated in the presence of a plasma sample positive for the respective anti-HLA antibody. A neutralization assay was also performed using purified immunoglobulin G (IgG) from two plasma samples, and two plasma samples were investigated in the presence of complement. The prerequisite for anti-HLA antibody-mediated neutralization is incorporation of HLA proteins by HIV-1. Therefore, the extent of incorporation of HLA proteins by the primary HIV-1 isolate was estimated. The ratios of HLA class I protein to HIV-1 capsid (p24) protein cultured in the PBMCs of two healthy individuals were 0.017 and 0.054. These ratios suggested that the HIV-1 strain used in the assay incorporated more HLA proteins than gp160 trimers. Anti-HLA antibody-positive plasma was found to contain antibodies that specifically reacted to HIV-1 carrying cognate HLA alleles. However, incubation of HIV-1 with anti-HLA antibody- positive plasma or purified IgG did not show a reduction in viral infectivity. HIV-1-neutralizing activity was also not detected in the presence of complement. This study shows that HIV-1 primary isolates cultured in PBMCs contain significant amounts of HLA proteins. However, the binding of antibodies to those HLA proteins does not mediate a reduction in viral infectivity.     

Neutralizing antibodies to an immunodominant envelope sequence do not prevent gp120 binding to CD4.

Animals immunized with the human immunodeficiency virus type 1 gp160 glycoprotein or certain recombinant envelope components develop potent virus-neutralizing activity. This activity is principally due to antibodies directed toward a hypervariable region of gp120 between cysteine residues 302 and 337 and is virus isolate specific. These antisera, as well as two neutralizing monoclonal antibodies directed against the same hypervariable sequence, do not appreciably block gp120 from binding CD4. In contrast, serum samples from infected humans possess high titers of antibodies that block gp120-CD4 binding; these titers approximately correlate with the serum neutralization titers. Our results suggest that there are at least two targets on the envelope glycoprotein for virus neutralization. The target responsible for the broader neutralizing activity of human serum may be a conserved region of gp120 involved in CD4 binding. The antibodies directed at the hypervariable region of the envelope inhibit a different step in virus infection which is subsequent to receptor binding. The extent to which these two different epitopes of gp120 may be involved in protection against human immunodeficiency virus infection is discussed.     

Analysis of Neutralization Specificities in Polyclonal Sera Derived from Human Immunodeficiency Virus Type 1-Infected Individuals

During human immunodeficiency virus type 1 (HIV-1) infection, patients develop various levels of neutralizing antibody (NAb) responses. In some cases, patient sera can potently neutralize diverse strains of HIV-1, but the antibody specificities that mediate this broad neutralization are not known, and their elucidation remains a formidable challenge. Due to variable and nonneutralizing determinants on the exterior envelope glycoprotein (Env), nonnative Env protein released from cells, and the glycan shielding that assembles in the context of the quaternary structure of the functional spike, HIV-1 Env elicits a myriad of binding antibodies. However, few of these antibodies can neutralize circulating viruses. We present a systematic analysis of the NAb specificities of a panel of HIV-1-positive sera, using methodologies that identify both conformational and continuous neutralization determinants on the HIV-1 Env protein. Characterization of sera included selective adsorption with native gp120 and specific point mutant variants, chimeric virus analysis, and peptide inhibition of viral neutralization. The gp120 protein was the major neutralizing determinant for most sera, although not all neutralization activity against all viruses could be identified. In some broadly neutralizing sera, the gp120-directed neutralization mapped to the CD4 binding region of gp120. In addition, we found evidence that regions of the gp120 coreceptor binding site may also be a target of neutralizing activity. Sera displaying limited neutralization breadth were mapped to the immunogenic V3 region of gp120. In a subset of sera, we also identified NAbs directed against the conserved, membrane-proximal external region of gp41. These data allow a more detailed understanding of the humoral responses to the HIV-1 Env protein and provide insights regarding the most relevant targets for HIV-1 vaccine design.     

Analysis of neutralizing epitopes on foot-and-mouth disease virus.

For the investigation of the antigenic determinant structure of foot-and-mouth disease virus (FMDV), neutralizing monoclonal antibodies (MAbs) against complete virus were characterized by Western blot (immunoblot), enzyme immunoassay, and competition experiments with a synthetic peptide, isolated coat protein VP1, and viral particles as antigens. Two of the four MAbs reacted with each of these antigens, while the other two MAbs recognized only complete viral particles and reacted only very poorly with the peptide. The four MAbs showed different neutralization patterns with a panel of 11 different FMDV strains. cDNA-derived VP1 protein sequences of the different strains were compared to find correlations between the primary structure of the protein and the ability of virus to be neutralized. Based on this analysis, it appears that the first two MAbs recognized overlapping sequential epitopes in the known antigenic site represented by the peptide, whereas the two other MAbs recognized conformational epitopes. These conclusions were supported and extended by structural analyses of FMDV mutants resistant to neutralization by an MAb specific for a conformational epitope. These results demonstrate that no amino acid exchanges had occurred in the primary antigenic site of VP1 but instead in the other coat proteins VP2 and VP3, which by themselves do not induce neutralizing antibodies.