B epitopes and selection pressures in feline immunodeficiency virus envelope glycoproteins.

In order to map linear B epitopes in feline immunodeficiency virus (FIV) envelope glycoproteins (Env), a random library of FIV Env polypeptides fused to beta-galactosidase and expressed in Escherichia coli was screened by using sera from experimentally FIV-infected cats. We mapped five antibody-binding domains in the surface envelope glycoprotein (SU1 to SU5) and four in the transmembrane envelope glycoprotein (TM1 to TM4). Immunological analysis with 48 serum samples from naturally or experimentally infected cats of diverse origins revealed a broad group reactivity for epitopes SU2, TM2, and TM3, whereas SU3 appeared as strictly type specific. To study selection pressures acting on the identified immunogenic domains, we analyzed structural constraints and distribution of synonymous and nonsynonymous mutations (amino acids unchanged or changed). Two linear B epitopes (SU3 and TM4) appeared to be submitted to positive selection for change, a pattern of evolution predicting their possible involvement in antiviral protection. These experiments provide a pertinent choice of oligopeptides for further analysis of the protective response against FIV envelope glycoproteins, as a model to understand the role of antibody escape in lentiviral persistence and to design feline AIDS vaccines.     

Antigenic variation of neutralization-sensitive epitopes of caprine arthritis-encephalitis lentivirus during persistent arthritis.

Caprine arthritis-encephalitis virus (CAEV), a naturally occurring lentivirus of goats, causes disease characterized by virus persistence and recurrent arthritis. These studies demonstrate in vitro neutralization of CAEV infectivity by serum from goats infected with CAEV. Serum neutralizing activity was not detectable until 10 to 36 months postinfection, and titers were relatively low (less than or equal to 1:8). Serum neutralization was caused by antibody and was virus specific. Antigenic variants of CAEV were isolated from cell-free joint fluid of arthritic goats 9 to 18 months postinfection. The delayed appearance of neutralizing antibody and the subsequent development of antigenic variants may promote CAEV persistence in vivo and provide a stimulus for recurrent arthritis.     

Characterization of CD4+ T helper cell fine specificity to the envelope glycoproteins of simian immunodeficiency virus

Virus-specific CD4+ T cells (Th) play a crucial role in the control of lentiviral replication. To better understand the epitope-specificity of CD4+ Th repertoire to the envelope glycoprotein (Env) of simian immunodeficiency virus (SIV), we analyzed Th responses to 20-mer overlapping Env peptides in eight genetically heterogeneous macaques chronically infected with live attenuated SIV. A set of 19 'broadly reactive' Th peptide-epitopes was defined from the distinct sets of responder peptides for individual macaques. The majority of broadly reactive peptide-epitopes (14 of 19) were uniformly distributed on the transmembrane (TM) domain of Env. Only five broadly reactive responder peptides localized to the surface domain (SU) of Env, and they were all confined to two non-glycosylated regions towards its carboxyl-terminus. This first comprehensive report of Env peptide-specific Th responses associated with attenuated SIV vaccine immunity indicates a profound influence of glycosylation on the development of Th responses and has important implications for acquired immunodeficiency syndrome (AIDS) vaccine development.     

Host Range of Small-Ruminant Lentivirus Cytopathic Variants Determined with a Selectable Caprine Arthritis- Encephalitis Virus Pseudotype System

The small-ruminant lentiviruses ovine maedi-visna virus (MVV) and caprine arthritis-encephalitis virus (CAEV) cause encephalitis, progressive pneumonia, arthritis, and mastitis in sheep and goats. Icelandic MVV strains, which are lytic in tissue culture, have a wide species distribution of functional receptors, which includes human cells. In contrast, functional receptors for the nonlytic CAEV CO are absent from human cells. To determine if the wide species distribution of functional receptors is a common property of MVV strains or related to cytopathic phenotype, we tested the infectivity of viruses pseudotyped with the envelope glycoproteins of MVV K1514, CAEV CO, and lytic and nonlytic North American MVV strains to cells of different species. Replication-defective CAEV proviral constructs lacking the env, tat, and vif genes and carrying the neomycin phosphotransferase gene in the vif-tat region were developed for the infectivity assays. Cotransfection of human 293T cells with these proviral constructs and plasmids expressing CAEV, MVV, or vesicular stomatitis virus envelope glycoproteins produced infectious pseudotyped virus which induced resistance of infected cells to G418. Using these pseudotypes, we confirmed the wide species distribution of Icelandic MVV receptors and the narrow host range of CAEV. However, functional receptors for the two North American MVV strains tested, unlike the Icelandic MVV and similar to CAEV, were limited to cells of ruminant species, regardless of cytopathic phenotype. The results indicate a differential receptor recognition by MVV strains which is unrelated to cytopathic phenotype.     

Gag protein epitopes recognized by CD4(+) T-helper lymphocytes from equine infectious anemia virus-infected carrier horses

Antigen-specific T-helper (Th) lymphocytes are critical for the development of antiviral humoral responses and the expansion of cytotoxic T lymphocytes (CTL). Identification of relevant Th lymphocyte epitopes remains an important step in the development of an efficacious subunit peptide vaccine against equine infectious anemia virus (EIAV), a naturally occurring lentivirus of horses. This study describes Th lymphocyte reactivity in EIAV carrier horses to two proteins, p26 and p15, encoded by the relatively conserved EIAV gag gene. Using partially overlapping peptides, multideterminant and possibly promiscuous epitopes were identified within p26. One peptide was identified which reacted with peripheral blood mononuclear cells (PBMC) from all five EIAV-infected horses, and three other peptides were identified which reacted with PBMC from four of five EIAV-infected horses. Four additional peptides containing both CTL and Th lymphocyte epitopes were also identified. Multiple epitopes were recognized in a region corresponding to the major homology region of the human immunodeficiency virus, a region with significant sequence similarity to other lentiviruses including simian immunodeficiency virus, puma lentivirus, feline immunodeficiency virus, Jembrana disease virus, visna virus, and caprine arthritis encephalitis virus. PBMC reactivity to p15 peptides from EIAV carrier horses also occurred. Multiple p15 peptides were shown to be reactive, but not all infected horses had Th lymphocytes recognizing p15 epitopes. The identification of peptides reactive with PBMC from outbred horses, some of which encoded both CTL and Th lymphocyte epitopes, should contribute to the design of synthetic peptide or recombinant vector vaccines for EIAV.     

Variability and immunogenicity of caprine arthritis-encephalitis virus surface glycoprotein

The complete surface glycoprotein (SU) nucleotide sequences of three French isolates of caprine arthritis-encephalitis virus (CAEV) were determined and compared with those of previously described isolates: three American isolates and one French isolate. Phylogenetic analyses revealed the existence of four distinct and roughly equidistant evolutionary CAEV subtypes. Four conserved and five variable domains were identified in the SU. The fine specificities of antibodies produced against these domains during natural infection were examined using a pepscan analysis. Nine immunogenic segments were delineated throughout the conserved and variable domains of SU, two of them corresponding to conserved immunodominant epitopes. Antigenic determinants which may be involved in the immunopathogenic process induced by CAEV were identified. These results also provide sensitive and specific antigen peptides for the serological detection and differentiation of CAEV and visna/maedi virus infections.     

Structure and genetic variability of envelope glycoproteins of two antigenic variants of caprine arthritis-encephalitis lentivirus.

To define the structure of the caprine arthritis-encephalitis virus (CAEV) env gene and characterize genetic changes which occur during antigenic variation, we sequenced the env genes of CAEV-63 and CAEV-Co, two antigenic variants of CAEV defined by serum neutralization. The deduced primary translation product of the CAEV env gene consists of a 60- to 80-amino-acid signal peptide followed by an amino-terminal surface protein (SU) and a carboxy-terminal transmembrane protein (TM) separated by an Arg-Lys-Lys-Arg cleavage site. The signal peptide cleavage site was verified by amino-terminal amino acid sequencing of native CAEV-63 SU. In addition, immunoprecipitation of [35S]methionine-labeled CAEV-63 proteins by sera from goats immunized with recombinant vaccinia virus expressing the CAEV-63 env gene confirmed that antibodies induced by env-encoded recombinant proteins react specifically with native virion SU and TM. The env genes of CAEV-63 and CAEV-Co encode 28 conserved cysteines and 25 conserved potential N-linked glycosylation sites. Nucleotide sequence variability results in 62 amino acid changes and one deletion within the SU and 34 amino acid changes within the TM.     

Detailed mapping of the antigenicity of the surface unit glycoprotein of equine infectious anemia virus by using synthetic peptide strategies.

We describe here a detailed analysis of the antigenic determinants of the surface unit glycoprotein (gp90) of equine infectious anemia virus (EIAV), using a comprehensive panel of synthetic peptides in enzyme-linked immunosorbent assays with immune serum from naturally and experimentally infected horses and with a panel of gp90-specific neutralizing and nonneutralizing monoclonal antibodies. The results of these studies identify immunoreactive segments throughout the conserved and variable domains of gp90 but localize immunodominant (100% reactivity) determinants to the amino and carboxyl termini of the glycoprotein molecule. Analysis of peptide reactivities with longitudinal serum samples taken from experimentally infected ponies revealed that antibody responses to conserved B-cell determinants appeared earlier and at higher titers than do antibodies specific for determinants contained in the variable domain of gp90. These observations suggest an evolution of antibody responses in EIAV-infected ponies that may correspond to the establishment of immunological control of virus replication and disease routinely observed in EIAV infections. In addition, the mapping of monoclonal antibody epitopes to peptides of 9 to 12 amino acids demonstrated that all of the neutralizing epitopes are located in the variable domain of gp90. The arrangement of neutralizing epitopes and critical structural considerations suggest that EIAV gp90 contains a principal neutralizing domain similar to the V3 loop of human immunodeficiency virus type 1. These antigenic analyses provide an important foundation for further analyzing the protective immune response generated during persistent EIAV infections and also provide potential peptide substrates for diagnostic assays and for vaccine strategies.     

The Humoral Immune Response to Human T-Cell Lymphotropic Virus Type 1 Envelope Glycoprotein gp46 Is Directed Primarily against Conformational Epitopes

Individuals infected with human T-cell lymphotropic virus type 1 (HTLV-1) develop a robust immune response to the surface envelope glycoprotein gp46 that is partially protective. The relative contribution of antibodies to conformation-dependent epitopes, including those mediating virus neutralization as part of the humoral immune response, is not well defined. We assess in this report the relationship between defined linear and conformational epitopes and the antibodies elicited to these domains. First, five monoclonal antibodies to linear epitopes within gp46 were evaluated for their ability to abrogate binding of three human monoclonal antibodies that inhibit HTLV-1-mediated syncytia formation and recognize conformational epitopes. Binding of antibodies to conformational epitopes was unaffected by antibodies to linear epitopes throughout the carboxy-terminal half and central domain of HTLV-1 gp46. Second, an enzyme-linked immunoadsorbent assay was developed and used to measure serum antibodies to native and denatured gp46 from HTLV-1-infected individuals. In sera from infected individuals, reactivity to denatured gp46 had an average of 15% of the reactivity observed to native gp46. Third, serum antibodies from 24 of 25 of HTLV-1-infected individuals inhibited binding of a neutralizing human monoclonal antibody, PRH-7A, to a conformational epitope on gp46 that is common to HTLV-1 and -2. Thus, antibodies to conformational epitopes comprise the majority of the immune response to HTLV-1 gp46, and the epitopes recognized by these antibodies do not appear to involve sequences in previously described immunodominant linear epitopes.     

Caprine arthritis encephalitis virus dysregulates the expression of cytokines in macrophages.

Caprine arthritis encephalitis virus (CAEV) is a lentivirus of goats that leads to chronic mononuclear infiltration of various tissues, in particular, the radiocarpal joints. Cells of the monocyte/macrophage lineage are the major host cells of CAEV in vivo. We have shown that infection of cultured goat macrophages with CAEV results in an alteration of cytokine expression in vitro. Constitutive expression of interleukin 8 (IL-8) and monocyte chemoattractant protein 1 (MCP-1) was increased in infected macrophages, whereas transforming growth factor beta1 (TGF-beta1) mRNA was down-regulated. When macrophages were infected with a CAEV clone lacking the trans-acting nuclear regulatory gene tat, IL-8 and MCP-1 were also increased. No significant differences from cells infected with the wild-type clone were observed, suggesting that Tat is not required for the increased expression of IL-8 and MCP-1 in infected macrophages. Furthermore, infection with CAEV led to an altered pattern of cytokine expression in response to lipopolysaccharide (LPS), heat-killed Listeria monocytogenes plus gamma interferon, or fixed cells of Staphylococcus aureus Cowan I. In infected macrophages, tumor necrosis factor alpha, IL-1beta, IL-6, and IL-12 p40 mRNA expression was reduced in response to all stimuli tested whereas changes in expression of granulocyte-macrophage colony-stimulating factor depended on the stimulating agent. Electrophoretic mobility shift assays demonstrated that, in contrast to effects of human immunodeficiency virus infection of macrophages, CAEV infection had no effect on the level of constitutive nuclear factor-kappaB (NF-kappaB) activity or on the level of LPS-stimulated NF-kappaB activity, suggesting that NF-kappaB is not involved in altered regulation of cytokine expression in CAEV-infected cells. In contrast, activator protein 1 (AP-1) binding activity was decreased in infected macrophages. These data show that CAEV infection may result in a dysregulation of expression of cytokines in macrophages. This finding suggests that CAEV may modulate the accessory functions of infected macrophages and the antiviral immune response in vivo.     

Overcoming T cell tolerance to the hepatitis B virus surface antigen in hepatitis B virus-transgenic mice

The sequence of the hepatitis B virus (HBV) major envelope (Env) protein (ayw subtype) was scanned for the presence of H-2(d,b) motifs. Following binding and immunogenicity testing, two new H-2(d)-restricted epitopes (Env.362 and Env.364) were identified. These epitopes induced CTLs capable of recognizing naturally processed HBV-Env, but were apparently generated with lower efficiency than the previously defined dominant Env.28 epitope. Next, HBV-transgenic mice that express all of the HBV proteins and produce fully infectious particles were immunized with a mixture of lipopeptides encompassing the Env.28, Env.362, and Env.364 epitopes. Significant CTL responses were obtained, but they had no effect on viral replication in the liver, nor did they induce an inflammatory liver disease. However, in adoptive transfer experiments, CTL lines generated from the HBV-transgenic mice following immunization were able to inhibit viral replication in vivo without causing hepatitis. This is in contrast to CTL lines derived from nontransgenic mice that displayed both antiviral and cytopathic effects, presumably because they displayed higher avidity for the viral epitopes than the transgenic CTLs. These results suggest that T cell tolerance to HBV can be broken with appropriate immunization but the magnitude and characteristics of the resultant T cell response are significantly different from the response in HBV-naive individuals since their antiviral potential is stronger than their cytotoxic potential. This has obvious implications for immunotherapy of chronic HBV infection.     

Peptide immunogen mimicry of putative E1 glycoprotein-specific epitopes in hepatitis C virus.

Hepatitis C virus (HCV) accounts for most cases of acute and chronic non-A and non-B hepatitis with serious consequences that may lead to hepatocellular carcinoma. The putative envelope glycoproteins (E1 and E2) of HCV probably play a role in the pathophysiology of the virus. In order to map the immunodominant domains of the E1 glycoprotein, two epitopes from amino acid residues 210 to 223 (P1) and 315 to 327 (P2) were predicted from the HCV sequence. Immunization of mice with the synthetic peptides conjugated to bovine serum albumin induced an antibody response, and the antisera immunoprecipitated the E1 glycoprotein (approximately 33 kDa) of HCV expressed by recombinant vaccinia virus. A panel of HCV-infected human sera was also tested with the synthetic peptides by enzyme-linked immunosorbent assay for epitope-specific responses. Of 38 infected serum samples, 35 (92.1%) demonstrated a spectrum of reactivity to the P2 peptide. On the other hand, only 17 of 38 (44.7%) serum samples were reactive to the P1 peptide. Strains of HCV exhibit a striking genomic diversity. The predicted P1 epitope showed localization in the sequence-variable region, and the P2 epitope localized in a highly conserved domain. Results from this study suggest that the E1 glycoprotein of HCV contains at least two potential antigenic epitopes. Synthetic peptides corresponding to these epitopes and antisera to these peptides may serve as the monospecific immunological reagents to further determine the role of E1 glycoprotein in HCV infection.     

Variable Constraints on the Principal Immunodominant Domain of the Transmembrane Glycoprotein of Human Immunodeficiency Virus Type 1

Lentiviruses have in their transmembrane glycoprotein (TM) a highly immunogenic structure referred to as the principal immunodominant domain (PID). The PID forms a loop of 5 to 7 amino acids between two conserved cysteines. Previous studies showed that envelope (Env) glycoprotein functions of feline immunodeficiency virus (FIV) could be retained after extensive mutation of the PID loop sequence, in spite of its high conservation. In order to compare Env function in different lentiviruses, either random mutations were introduced in the PID loop sequence of human immunodeficiency virus type 1 (HIV-1) or the entire HIV-1 PID loop was replaced by the corresponding PID loop of FIV or simian immunodeficiency virus (SIV). In the macrophage-tropic HIV-1 ADA Env, mutations impaired the processing of the gp160 Env precursor, thereby abolishing viral infectivity. However, 6 of the 108 random Env mutants that were screened retained the capacity to induce cell membrane fusion. The SIV and FIV sequences and five random mutations were then introduced in the context of T-cell-line-adapted HIV-1 LAI which, although phenotypically distant from HIV-1 ADA, has an identical PID loop sequence. In contrast to the situation for HIV-1 ADA mutants, the cleavage of the Env precursor was unaffected in most HIV-1 LAI mutants. Such mutations, however, resulted in increased shedding of the gp120 surface glycoprotein (SU) from the gp41 TM. The HIV-1 LAI Env mutants showed high fusogenic efficiency. Three Env mutants retained the capacity to mediate virus entry in target cells, although less efficiently than the wild-type Env, and allowed the reconstitution of infectious molecular clones. These results indicated that in HIV-1, like FIV, the conserved PID sequence can be changed without impairing Env function. However, functional constraints on the PID of HIV-1 vary depending on the structural context of Env, presumably in relation to the role of the PID in the interaction of the SU and TM subunits and the stability of the Env complex.     

Characterization of the viral O-glycopeptidome: a novel tool of relevance for vaccine design and serodiagnosis

Viral envelope proteins mediate interactions with host cells, leading to internalization and intracellular propagation. Envelope proteins are glycosylated and are known to serve important functions in masking host immunity to viral glycoproteins. However, the viral infectious cycle in cells may also lead to aberrant glycosylation that may elicit immunity. Our knowledge of immunity to aberrant viral glycans and glycoproteins is limited, potentially due to technical limitations in identifying immunogenic glycans and glycopeptide epitopes. This work describes three different complementary methods for high-throughput screening and identification of potential immunodominant O-glycopeptide epitopes on viral envelope glycoproteins: (i) on-chip enzymatic glycosylation of scan peptides, (ii) chemical glycopeptide microarray synthesis, and (iii) a one-bead-one-compound random glycopeptide library. We used herpes simplex virus type 2 (HSV-2) as a model system and identified a simple O-glycopeptide pan-epitope, (501)PPA(GalNAc)TAPG(507), on the mature gG-2 glycoprotein that was broadly recognized by IgG antibodies in HSV-2-infected individuals but not in HSV-1-infected or noninfected individuals. Serum reactivity to the extended sialyl-T glycoform was tolerated, suggesting that self glycans can participate in immune responses. The methods presented provide new insight into viral immunity and new targets for immunodiagnostic and therapeutic measures.     

Continuous epitopes of the human immunodeficiency virus type 1 (HIV-1) transmembrane glycoprotein and reactivity of human sera to synthetic peptides representing various HIV-1 isolates.

Immunoreactive regions of human immunodeficiency virus type 1 (HIV-1) gp41 were mapped by reacting HIV-1 antibody-positive human sera with overlapping synthetic peptides which covered the transmembrane protein. Three immunoreactive domains were identified, and five different and partially overlapping epitopes recognized by HIV-1-positive human sera were found within one immunodominant region. The effect on antibody recognition after single amino acid substitutions within one defined epitope was also studied. The reactivity of various HIV-1-positive sera to synthetic peptides with amino acid substitutions representing known isolates suggests an important substitution in the major epitope of African HIV-1 strains.     

Characterization of immunoreactive epitopes of the HIV-1 p41 envelope protein using fusion proteins synthesized in Escherichia coli

We have identified several immunoreactive epitopes on the human immunodeficiency virus (HIV) type 1 transmembrane envelope protein by synthesizing various regions of the protein as fusions to the trpE gene in Escherichia coli. Ten fusion clones which expressed overlapping peptides were found to contain epitopes reactive with antibodies in sera of North American (NAm) and West African (WAf) patients with acquired immune deficiency syndrome (AIDS). An immunodominant epitope which reacted with all HIV-infected patients' sera was mapped to a 51-amino acid sequence in the N terminus of p41. A novel epitope was also identified in the C terminus of p41 which was reactive with 41% and 48% of the sera tested from NAm and WAf, respectively. In addition, several minor epitopes were identified. We observed that sera from WAf reacted more strongly to minor HIV-1 p41 epitopes than did sera from similarly infected individuals in NAm. We also report on the detection of antibodies from patients with HIV-2 infection in WAf which cross react with HIV-1 p41 recombinant envelope antigens.     

The recognition of a viral antigenic moiety by class I MHC-restricted cytolytic T lymphocytes is limited by the availability of the endogenously processed antigen.

The transmembrane hydrophobic domain of the type A influenza A/JAPAN/305/57 (H2N2) hemagglutinin (HA) contains an immunodominant site encompassing amino acids 523-545 (J523-545) recognized by class I MHC-restricted cytolytic T lymphocytes (CTL). Class I CTL of two fine specificity subsets map to this transmembrane (TM) site. One of these CTL subpopulations is subtype specific. These T lymphocytes recognize the site generated during infection of target cells with A/JAPAN/305/57 virus (H2N2) but not target cells expressing the comparable TM site of the influenza A/PR/8/34 virus (H1N1) hemagglutinin (P527-549) after infection with this virus. The other CTL subpopulation is cross-reactive and recognizes the TM site of the A/JAPAN/305/57 HA and the A/PR/8/34 HA with similar efficiency. Analyses of the critical amino acids in the TM site necessary for CTL recognition with the use of synthetic peptides unexpectedly revealed reactivity for the A/PR/8 HA TM site by subtype-specific CTL. This reactivity was only observed with truncated peptides corresponding to a limited portion of the A/PR/8 HA TM site but also required peptide concentrations greater than 10(-7) M. These results suggested either that the endogenously processed A/PR/8 HA TM site generated during infection was larger than the site defined by the truncated cross-reactive peptides or that the concentration of endogenously processed TM site produced during infection was limiting. To distinguish between these possibilities, we expressed in target cells synthetic minigenes encoding only the portion of the A/PR/8 HA transmembrane sites defined by the synthetic peptides. Unlike the peptides, the "preprocessed" endogenous minigene products were not recognized by subtype-specific CTL. These data suggest that the level of available endogenously processed Ag rather than selectivity in the site of fragmentation of newly synthesized Ag may play a critical role in determining whether the complex of the antigenic moiety and class I MHC is efficiently presented to and recognized by class I CTL.     

Detailed analysis of CD4+ Th responses to envelope and Gag proteins of simian immunodeficiency virus reveals an exclusion of broadly reactive Th epitopes from the glycosylated regions of envelope

Ag-specific CD4(+) Th cells play a key role in the development, maturation, and maintenance of pathogen-specific humoral and cellular immune responses. To define the fine specificity of broadly reactive Th responses associated with mature immunity in a lentiviral system, we analyzed peptide-specific Th responses in eight macaques chronically infected with a reference live attenuated SIV at 12-14 mo postinoculation. All macaques had stable immunocompetent Th cells at the time of analysis, and a unique array of Th responses to 20-mer overlapping peptides from envelope (Env) and Gag was identified for each macaque, which were then used to define a set of 31 broadly reactive peptide epitopes. Only 5 of the 31 broadly reactive Th epitope peptides mapped to the surface (SU) domain of Env. Interestingly, these were all confined to two conserved nonglycosylated regions toward the carboxyl terminus of SU, suggesting a structural influence of glycosylation on development of Th responses. Gag and the Env transmembrane proteins contained the majority of broadly reactive peptide epitopes (12 and 14 peptides, respectively), which were uniformly distributed throughout their sequence. This study defines for the first time broadly reactive Th epitope peptides of SIV Env and Gag proteins that are associated with enduring broadly protective vaccine immunity to attenuated SIV, which may be used for the design and evaluation of experimental vaccines. Moreover, the data suggest that extensive glycosylation of SU may provide yet another immune escape mechanism developed by lentiviruses to restrict the breadth of Th repertoire to SU, a major immunologically exposed protein of the virus.