Characterization of simian-human immunodeficiency virus envelope glycoprotein epitopes recognized by neutralizing antibodies from infected monkeys

We characterized human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein epitopes recognized by neutralizing antibodies from monkeys recently infected by molecularly cloned simian-human immunodeficiency virus (SHIV) variants. The early neutralizing antibody response in each infected animal was directed mainly against a single epitope. This primary neutralizing epitope, however, differed among individual monkeys infected by identical viruses. Two such neutralization epitopes were determined by sequences in the V2 and V3 loops of the gp120 envelope glycoprotein, while a third neutralization epitope, apparently discontinuous, was determined by both V2 and V3 sequences. These results indicate that the early neutralizing antibody response in SHIV-infected monkeys is monospecific and directed against epitopes composed of the gp120 V2 and V3 variable loops.     

Conserved N-linked oligosaccharides of the C-terminal portion of human immunodeficiency virus type 1 gp120 and viral susceptibility to neutralizing antibodies

We have constructed a mutated infectious HIV variant lacking the signals for addition of three N-linked glycans situated in the V4, C4 and V5 regions of HIV gp120. When comparing mutated virus with wildtype virus we found essentially no differences in the phenotypic characteristics of the two viruses except for the expected electrophoretic mobility shift of radioimmuno-precipitated mutated gp120, resulting from the missing N-glycans. Thus, the infectivity titer and the capacity to induce syncytia were similar for the two viruses. The sensitivity of mutant and wildtype virus to a number of neutralizing agents was determined. As expected, the mutant virus was significantly less sensitive to neutralization by Con A, with affinity for the N-glycans eliminated. We found, however, that antibodies to the V3 loop and sCD4 neutralized wild-type virus as efficiently as mutant virus, whereas 2G12, a monoclonal antibody, binding to a discontinuous neutralization epitope, and GP13, binding to the CD4-binding domain, neutralized wildtype virus better than mutant virus. Altogether the data suggest that the three conserved N-linked glycans, despite their location in immediate association with the CD4-binding domain, which is an important neutralization epitope, are not essential for virus replication in cell culture and they are not engaged in shielding neutralization epitopes of gp120 from neutralizing antibodies. However, the glycans evidently influence the three-dimensional conformation of gp120, since their presence increases the availability of the neutralization epitope of 2G12.     

Live attenuated simian immunodeficiency virus (SIV)mac in macaques can induce protection against mucosal infection with SIVsm

OBJECTIVE: To investigate whether vaccination of macaques with attenuated simian immunodeficiency virus (SIV)macC8 could induce long-term protective immunity against rectal exposure to SIVsm and intravenous exposure to the more divergent HIV-2. DESIGN AND METHODS: Eight months after vaccination with live attenuated SIVmacC8, four cynomolgus monkeys were challenged with SIVsm intrarectally and another four vaccinated monkeys were challenged with HIV-2 intravenously. Sixteen months after SIVmacC8 vaccination, another two monkeys were challenged with SIVsm across the rectal mucosa. Two vaccinees shown to be protected against SIVsm were rechallenged 8 months after the first challenge. Ten naive animals were used as controls. Serum antigenaemia, virus isolation, antibody responses, cell-mediated immunity and CD4+ and CD8+ T-cell subpopulations were monitored. PCR-based assays were used to distinguish between virus populations. RESULTS: At the time of challenge, eight out of 10 vaccinees were PCR-positive for SIVmacC8 DNA but no virus could be isolated from peripheral blood mononuclear cells. After SIVsm challenge, three out of six vaccinees were repeatedly SIVsm PCR-negative. In one of the three infected monkeys, the challenge virus was initially suppressed but the monkey ultimately developed AIDS after increased replication of the pathogenic virus. Rechallenged monkeys remained protected. All HIV-2-challenged vaccinees became superinfected. All controls became infected with either SIVsm or HIV-2. At the time of challenge the vaccinees had neutralizing antibodies to SIVmac but no demonstrable cross-neutralizing antibodies to SIVsm or HIV-2. Titres of antigen-binding or neutralizing antibodies did not correlate with protection. Cytotoxic T-cell responses to SIV Gag/Pol and virus-specific T-cell proliferative responses were low. CONCLUSION: The live attenuated SIVmacC8 vaccine was able to induce long-term protection against heterologous intrarectal SIVsm challenge in a proportion of macaques but not against the more divergent HIV-2, which was given intravenously.     

Obstructive sleep apnea syndrome in the Community of Valencia: current situation, study of needs and future prospects]

Experimental simian varicella virus (SVV) infection of St. Kitts vervet monkeys was evaluated as an animal model to investigate human varicella-zoster virus (VZV) infections. During the incubation period, viremia disseminated infectious virus throughout the body via infected peripheral blood lymphocytes (PBLs). A vesicular skin rash in the inguinal area, and on the abdomen, extremities, and face appeared on day 7-10 postinfection. Necrosis and hemorrhage in lung and liver tissues from acutely infected monkeys were evident upon histologic analysis. Recovery from simian varicella was accompanied by a rise in the serum neutralizing antibody response to the virus. SVV latency was established in trigeminal ganglia of monkeys which resolved the acute infection. This study indicates that experimental SVV infection of St. Kitts vervets is a useful animal model to investigate SVV and VZV pathogenesis and to evaluate potential antiviral agents and vaccines.     

Broad cross-neutralizing activity in serum is associated with slow progression and low risk of transmission in primate lentivirus infections

Sera from human immunodeficiency virus type 1 and type 2 (HIV-1 and HIV-2)-infected humans were tested with autologous (from the same individual) and heterologous (from other individuals) virus isolates in a neutralization assay. Similarly, sera from experimentally simian immunodeficiency virus (SIVsm from sooty mangabey) or HIV-2SBL6669-infected cynomolgus macaques were tested for neutralizing activity against autologous and heterologous reisolates. In the neutralization assay, the virus dose ranged between 10-75 50% infectious dose (ID50), sera were used in five 2- or 4-fold dilutions, beginning with 1:20, and human peripheral blood mononuclear cells (PBMCs) served as target cells. The readout of the 7-day assay was a HIV-1 or HIV-2 antigen enzyme-linked immunosorbent assay (ELISA). Our results show that SIVsm-inoculated monkeys who develop early immunodeficiency lack serum neutralizing activity or develop a neutralizing antibody response with narrow specificity. Long survival is associated with the ability to neutralize several autologous and heterologous SIVsm reisolates. Infection of macaques with HIV-2SBL6669 did not cause disease within the 5 years observation time and elicited a broadly cross-reactive neutralizing antibody response, including neutralization of other, independently obtained, HIV-2 isolates. In HIV-1-infected humans, neutralizing antibodies can only be detected in up to 50% of cases. Neutralizing activity, whenever present, may show a broad specificity, that is, neutralization may occur across genetic subtypes. Presence of broadly cross-reactive neutralizing antibodies is associated with a lower risk of HIV-1 (subtype B) transmission both from mother to child and sexually from male to female. Unlike HIV-1 infection, serum neutralizing activity is regularly present in HIV-2 infection. In view of the differences between HIV-1 and HIV-2 pathogenesis, we suggest that an effective neutralizing antibody response may contribute to a delay in disease progression and to a decrease in risk of transmission.     

Single amino acid substitution in constant region 1 or 4 of gp120 causes the phenotype of a human immunodeficiency virus type 1 variant with mutations in hypervariable regions 1 and 2 to revert

The second major cysteine loop of human immunodeficiency virus type 1 envelope glycoprotein gp120 contains 5 to 11 consensus N-linked glycosylation sites, which is disproportionately higher than the number of such sites found in other regions of gp120. Amino acid substitutions introduced at three of six N-linked glycosylation sites in this region of an infectious molecular clone, HXB2, resulted in severe impairment of virus infectivity. Isolation and genetic characterization of a revertant of this mutant revealed an isoleucine-for-valine substitution at position 84 in constant region 1 and an isoleucine-for-methionine substitution at position 434 in constant region 4. Further mutational analysis indicated that either isoleucine substitution was sufficient to confer the revertant phenotype. These findings demonstrate that V1/V2 not only functionally interacts with C4, as previously reported, but also interacts with C1. The observation that compensatory changes do not involve regeneration of N-linked glycosylation sites in the second major cysteine loop suggests that replication of human immunodeficiency virus type 1 in vitro is independent of the presence of a disproportionate number of N-linked glycosylation sites within this loop.     

Identification and characterization of the glycoprotein E and I genes of canine herpesvirus

We have determined the sequence of the gE and gI genes of canine herpesvirus (CHV), DFD-6 strain. The gE ORF codes for a 522 a.a. polypeptide with a signal sequence at the amino-terminus and a trans-membrane domain at the carboxy-terminus. The gI ORF codes for a 259 a.a. polypeptide with a signal sequence but no trans-membrane domain. Comparison with another line of CHV indicated that the DFD-6 gI gene underwent a frame-shift mutation which caused the loss of the trans-membrane domain. Antibodies against the gE and gI polypeptides detected a 94 kDa gE and a broad band of gI (55-62 kDa) in DFD-6 infected cells, respectively. The precursor of DFD-6 gE is modified to the mature form by N-linked glycosylation only in the presence of gI. Together with the fact that the gI- mutant of DFD-6 produced smaller plaques, it is suggested that the truncated DFD-6 gI is functional. The precursor of DFD-6 gI is modified to the mature form by N-linked glycosylation only in the presence of gE.     

Congenital abnormalities in newborn calves after inoculation of pregnant cows with Akabane virus

A fresh isolate of Akabane virus was inoculated intravenously into 11 seronegative pregnant cows at 62 to 96 days of gestation. Two of the cows were slaughtered 18 days post-inoculation, and the fetuses were examined; the remaining cows were allowed to give birth. All the inoculated cows developed viremia and neutralizing antibody for the virus, indicating that the cows were actually infected with the virus, although fever or any other clinical abnormalities were not noted. The virus further infected the fetuses. This was proved by virus isolation in one of the two fetuses from the slaughtered cows, and polymyositis was noted in both fetuses. Six of seven calves born alive had anti-Akabane antibody in their precolostral sera, indicating that in utero infection with the virus took place in these calves. Some of the in utero-infected calves demonstrated congenital abnormalities such as cerebral defect, hydranencephaly, and arthrogryposis. These findings provide additional evidence that Akabane virus is the etiological agent of epizootic abortion and congenital arthrogryposis-hydranencephaly syndrome in cattle.     

Detection of neutralizing antibodies against human T-cell leukemia virus type 1 using a cell-free infection system and polymerase chain reaction

We have developed a cell-free infection system to titrate neutralizing antibodies against human T-cell leukemia virus type 1 (HTLV-1) using the polymerase chain reaction (PCR). S+L-CCC (8C) feline kidney or U-251 MG human glioma cells were infected with a cell-free culture supernatant derived from HTLV-1-infected c77 feline cells. DNA was extracted from 8C or U-251 MG cells after incubation for 24 hr and amplified by PCR. The c77 cell supernatant gave discrete bands, whereas those of HTLV-1-positive T cells did not. When the inocula were treated with HTLV-1 antibody-positive human sera or the monoclonal or polyclonal antibody against the peptide 190-199 of HTLV-1 envelope protein gp46, the subsequent formation of HTLV-1 proviral DNA was inhibited. We determined the titers of neutralizing antibodies by densitometrically scanning the intensity of the PCR bands. These titers correlated well with those determined by the plaque assay using a pseudotype of vesicular stomatitis virus bearing the envelope antigens of HTLV-1. At high serum concentrations, many seronegative samples markedly inhibited the plating of the HTLV-1 pseudotype whereas they barely affected results obtained by PCR. Thus, the c77-PCR system can detect neutralizing antibodies against HTLV-1 even at low titers.     

Neutralization of HIV-1 primary isolates by polyclonal and monoclonal human antibodies

To examine antibody-mediated neutralization of HIV-1 primary isolates in vitro, we tested sera and plasma from infected individuals against four clade B primary isolates. These isolates were analyzed further for neutralization by a panel of several human anti-HIV-1 mAb in order to identify the neutralizing epitopes of these viruses. Each of the HIV-1+ serum and plasma specimens tested had neutralizing activities against one or more of the four primary isolates. Of the three individual sera, one (FDA-2) neutralized all of the four isolates, while the other two sera were effective against only one virus. The pooled plasma and serum samples reacted broadly with these isolates. Based on the neutralizing activities of the mAb panel, each virus isolate exhibited a distinct pattern of reactivity, suggesting antigenic diversity among clade B viruses. Neutralizing epitopes were found in the V3 loop and CD4-binding domain of gp120, as well as near the transmembrane region (cluster II epitope) of gp41. A mAb directed to the cluster I epitope of gp41 near the immunodominant disulfide loop weakly neutralized one primary isolate. None of the mAb in the panel affected one primary isolate, US4, although this virus was sensitive to neutralization by some of the polyclonal antibody specimens. This isolate was also resistant to neutralization by a cocktail of 10 mAb, most of which individually inhibited at least one of the other three viruses tested. These results suggest that neutralizing activity for this latter virus is present in certain HIV-1+ sera/plasma, but is not exhibited by the mAb in the panel. Thus, effective neutralizing antibodies against primary isolates can be generated by humans upon exposure to HIV-1, but not all of these antigenic specificities are represented in a large panel of human anti-HIV-1 mAb.     

Env-independent protection induced by live, attenuated simian immunodeficiency virus vaccines

Live attenuated simian immunodeficiency viruses (SIV), such as nef deletion mutants, are the most effective vaccines tested in the SIV-macaque model so far. To modulate the antiviral immune response induced by live attenuated SIV vaccines, we had previously infected rhesus monkeys with a nef deletion mutant of SIV expressing interleukin 2 (SIV-IL2) (B. R. Gundlach, H. Linhart, U. Dittmer, S. Sopper, S. Reiprich, D. Fuchs, B. Fleckenstein, G. Hunsmann, S. Stahl-Hennig, and K. Uberla, J. Virol. 71:2225-2232, 1997). In the present study, SIV-IL2-infected macaques and macaques infected with the nef deletion mutant SIVDeltaNU were challenged with pathogenic SIV 9 to 11 months postvaccination. In contrast to the results with naive control monkeys, no challenge virus could be isolated from the SIV-IL2- and SIVDeltaNU-infected macaques. However, challenge virus sequences could be detected by nested PCR in some of the vaccinated macaques. To determine the role of immune responses directed against Env of SIV, four vaccinated macaques were rechallenged with an SIV-murine leukemia virus (MLV) hybrid in which the env gene of SIV had been functionally replaced by the env gene of amphotropic MLV. All vaccinated macaques were protected from productive infection with the SIV-MLV hybrid in the absence of measurable neutralizing antibodies, while two naive control monkeys were readily infected. Since the SIV-MLV hybrid uses the MLV Env receptor Pit2 and not CD4 and a coreceptor for virus entry, chemokine inhibition and receptor interference phenomena were not involved in protection. These results indicate that the protective responses induced by live attenuated SIV vaccines can be independent of host immune reactions directed against Env.     

Identification of the sites of N-linked glycosylation on the human calcium receptor and assessment of their role in cell surface expression and signal transduction

The human calcium receptor (hCaR) is a G-protein-coupled receptor containing 11 potential N-linked glycosylation sites in the large extracellular domain. The number of potential N-linked glycosylation sites actually modified, and the effect on cell surface expression and signal transduction of blocking glycosylation at these sites, was examined by site-directed mutagenesis. Asparagine residues of the consensus sequences (Asn-Xaa-Ser/Thr) for N-linked glycosylation were mutated to glutamine individually and in various combinations to disrupt the potential N-linked glycosylation sites in the context of the full-length receptor. The cDNA constructs were transiently transfected into HEK-293 cells lacking endogeneous hCaR, and expressed receptors were analyzed by mobility differences on immunoblots, glycosidase digestion, intact cell enzyme-linked immunoassay, and extracellular calcium-stimulated phosphoinositide hydrolysis assay. Immunoblot analyses and glycosidase digestion studies of the wild type versus mutant receptors demonstrate that, of the 11 potential sites for N-linked glycosylation, eight sites (Asn-90, -130, -261, -287, -446, -468, -488, and -541) are glycosylated; the three remaining sites (Asn-386, -400, and -594) may not be efficiently glycosylated in the native receptor. Sequential mutagenesis of multiple N-linked glycosylation sites and analyses by immunoblotting, immunofluorescence, biotinylation of cell surface proteins, and intact cell enzyme-linked immunoassay indicated that disruption of as few as three glycosylation sites impairs proper processing and expression of the receptor at the cell surface. Disruption of five glycosylation sites reduced cell surface expression by 50-90% depending on which five sites were disrupted. Phosphoinositide hydrolysis assay results for various glycosylation-defective mutant receptors in general correlated well with the level of cell surface expression. Our results demonstrate that among 11 potential N-linked glycosylation sites on the hCaR, eight sites are actually utilized; glycosylation of at least three sites is critical for cell surface expression of the receptor, but glycosylation does not appear to be critical for signal transduction.     

HIV-related musculoskeletal disease

Breast feeding is the major route of mother-to-child transmission of human T-cell leukemia virus type I (HTLV-I). Our experiments with rabbits have shown that passive immunization is capable of blocking cell-to-cell infection of HTLV-I by blood transfusion or breast feeding. In this study, sera were collected serially from 3 infants born to seropositive mothers and were tested for the presence of neutralizing antibody to vesicular stomatitis virus (HTLV-I) pseudotype as well as antibodies to viral structural proteins. There was a good correlation between neutralizing and viral antibody titers, both of which were detectable until 3-6 months after birth. Whether maternally transmitted neutralizing antibody is protective against perinatal infection of HTLV-I remains to be studied.     

Gas chromatography in express diagnosis of candidiasis and monitoring of antifungal therapy efficacy by D-arabinitol and mannose levels in pediatric patients]

Methods for preventing and treating Ebola virus hemorrhagic fever are not still available despite the fact that this virus have been studied for 20 years. Methods of immunization of the animals (sheep, goats) non-susceptible to Ebola virus with live virus preparations were developed to obtain the hyperimmune anti-Ebola virus sera required to have highly immune antivirus gamma-globulins. These methods made it possible to obtain the immune sera having high virus-neutralizing antibodies. Caprine immunoglobulins were obtained from sera by fractionation of immune sera by Kohn's method. The neutralization indices of the immunoglobulins obtained were at least Ig. When administered in the first hours of infection, the protective effect of these preparations was shown on guinea pigs infected with LD50 of the strain pathogenic to the animals. Preclinical trials of these immunoglobulins on laboratory animals and clinical trials on volunteers were performed. The preparation was used as a preventive agent when accidents took place at the laboratory working with Ebola virus. The similar preparation from equine sera having high neutralizing and protective properties was elaborated at the Virological Center, Microbiological Institute, Russian Ministry of Defense. Its prophylactic efficiency was also shown in infected gamadrias.     

Recombinant bacille Calmette-Guérin expressing the measles virus nucleoprotein protects infant rhesus macaques from measles virus pneumonia

Measles virus infection continues to be a major cause of infant mortality. There is a need for a measles vaccine that can be administered at birth in the presence of maternal neutralizing antibody. Infant rhesus monkeys were immunized with recombinant bacille Calmette-Guérin expressing the full-length measles virus nucleoprotein (BCG-N) and subsequently challenged with measles virus. Nucleoprotein-specific lymphocyte proliferative responses were detected in the absence of anti-N antibody after vaccination. Vaccination with BCG-N did not prevent systemic measles virus infection; however, there was a significant reduction of lung inflammation after challenge. Virus titers in lymph nodes were significantly lower, and the duration of nasopharyngeal viral shedding was shorter in some vaccinated monkeys after challenge. These results suggest that measles virus-specific T cells were primed by BCG-N vaccination and that they prevented virus-induced lung pathology.     

N-linked glycosylation of the human Ca2+ receptor is essential for its expression at the cell surface

The human Ca2+ receptor (hCaR) is a member of the superfamily of G protein-coupled receptors. Its large (approximately 600 residue) amino-terminal extracellular domain contains 9 potential N-linked glycosylation sites. Immunoblot of cell membranes derived from HEK-293 cells, stably transfected with the hCaR, showed two major immunoreactive bands of approximately 150 and 130 kDa, respectively. Complete digestion of the membranes with PN-glycosidase F yielded a single major immunoreactive band of approximately 115 kDa, confirming the presence of N-linked glycosylation. Treatment of these cells with tunicamycin, which blocks N-linked glycosylation, inhibited signal transduction in response to Ca2+. Flow cytometric analysis showed decreased expression of the hCaR on the cell membrane in tunicamycin-treated cells. Immunoblot of tunicamycin-treated cells showed a reduction in the amount of the 150-kDa band and conversion of the 130-kDa band to the presumptively nonglycosylated 115-kDa form. Tunicamycin treatment of cells, transfected with a mutant hCaR complementary DNA containing a nonsense codon at position 599 preceding the 1st transmembrane domain, blocked the secretion of a 95-kDa protein, representing the amino-terminal extracellular domain, into the medium. These results demonstrate that N-linked glycosylation is required for normal expression of the hCaR at the cell surface.     

From plant to patient: an ethnomedical approach to the identification of new drugs for the treatment of NIDDM

A micro-focus reduction neutralization test (mFRNT) was evaluated as an alternative test to the ordinary plaque reduction neutralization test (PRNT) for the determination of dengue virus and Japanese encephalitis virus neutralizing antibody responses in persons receiving dengue vaccine. The 2 tests were similar in terms of titres and ability to detect seroconversion. Although the neutralizing antibody titres obtained by mFRNT were slightly lower than those given by PRNT, the differences were less than two-fold, indicating than mFRNT was reliable. Reproducibility of mFRNT was confirmed by 10 replicate tests using the same control serum. Therefore, mFRNT may be useful in large-scale investigations of neutralizing antibody levels, for example, in young children forming part of an immunization programme; it can be performed quickly and is economical, requiring only a small volume of sera.     

Role of asparagine-linked oligosaccharides in protein folding, membrane targeting, and thyrotropin and autoantibody binding of the human thyrotropin receptor

The amino-terminal ectodomain of thyrotropin (TSH) receptor (TSHR) is heavily glycosylated with asparagine-linked (N-linked) oligosaccharides. The present studies were designed to evaluate how acquisition and processing of N-linked oligosaccharides play a role in the functional maturation of human TSHR. A glycosylation inhibitor tunicamycin, which inhibits the first step of N-linked glycosylation (acquisition of N-linked oligosaccharides), and a series of mutant Chinese hamster ovary (CHO)-Lec cells defective in the different steps of glycosylation processing were used. Inhibition of acquisition of N-linked oligosaccharides by tunicamycin treatment in CHO cells stably expressing TSHR produced nonglycosylated TSHR, which was totally nonfunctional. In contrast, all of the TSHRs synthesized in mutant CHO-Lec1, 2, and 8 cells (mannose-rich, sialic acid-deficient, and galactose-deficient oligosaccharides, respectively) bound TSH and produced cAMP in response to TSH with an affinity and an EC50 similar to those in TSHR expressed in parental CHO cells (CHO-TSHR; sialylated oligosaccharides). However, Lec1-TSHR and Lec2-TSHR were not efficiently expressed on the cell surface, whereas the expression levels of Lec8-TSHR and CHO-TSHR were essentially identical. All of the TSHRs expressed in CHO-Lec cells cleaved into two subunits. Finally, anti-TSHR autoantibodies from Graves' patients interacted with all of the TSHRs harboring different oligosaccharides to a similar extent. These data demonstrate that acquisition and processing of N-linked oligosaccharides of TSHR appear to be essential for correct folding in the endoplasmic reticulum and for cell surface targeting in the Golgi apparatus. We also show that complex type carbohydrates are not crucially involved in the interaction of TSHR with TSH and anti-TSHR autoantibodies.     

An improved enzyme linked immunosorbent assay for detection of anti-ranavirus antibodies in the serum of the giant toad (Bufo marinus)

An improved ranavirus antibody ELISA (R Ab ELISA) for the specific detection of anti-ranavirus antibodies in toad sera was developed. Sheep anti-epizootic haematopoietic necrosis virus (EHNV) was used as the antigen-capture antibody. EHNV was used as the antigen and sera from field and challenged toads were used to detect the virus. Rabbit anti-toad IgG and IgM were used to detect bound toad antibody. Pre-absorption of toad sera with a monoclonal antibody, raised against the 50 kDa EHNV protein, improved the specificity of the technique. A blocking ELISA, immunofluorescence and immuno-electron microscopy were used to confirm the validity of the ELISA. The assay has potential use in screening sera from Bufo marinus for the presence of antibodies against ranaviruses and to facilitate understanding of the humoral immunological response in toads during virus infection.