Biomedical subjects

R C Montelaro

Publications and source records attributed to R C Montelaro.

At least 19 recordsLinked to original sources

PubMed1992

Expression of the protease gene of equine infectious anemia virus in Escherichia coli: formation of the mature processed enzyme and specific cleavage of the gag precursor.

A 620-bp Bg/II restriction fragment containing the putative protease coding sequence from equine infectious anemia virus (EIAV) proviral DNA was cloned and expressed in E. coli as a Pol precursor protein. In contrast to the 25-kDa fusion protein predicted from the expressed pol sequence, a protein of approximately 10 kDa was generated by apparent autocatalytic processing of the Pol precursor. This mature processed protein was detected in transformed cells using an antisera raised against synthetic peptide from the conserved carboxyl-terminal segment of the predicted EIAV protease coding sequence. Coexpression of this protein with a 35-kDa EIAV Gag-precursor fusion protein resulted in the specific proteolytic processing of the precursor as shown by formation of p26, the major capsid protein of EIAV.

Cloning, Molecular↗

PubMed1992

Identification of broadly reactive continuous antigenic determinants of simian immunodeficiency virus glycoproteins.

The env polyprotein sequences of several simian immunodeficiency virus (SIV) isolates were analyzed using computer algorithms designed to predict immunologically reactive protein segments. Peptides corresponding to predicted epitopes were synthesized and employed in peptide enzyme-linked immunosorbent assay (ELISA) screening of serum panels from experimentally infected macaques, as well as naturally infected, asymptomatic mangabeys and African green monkeys. Several of the peptides are recognized by a high percentage of antisera from each panel of monkeys indicating that they represent group-specific antigenic determinants of SIV. Several type-specific determinants also were identified. These peptides may be a useful tool for studying the kinetics of SIV glycoprotein-specific immune responses produced by infected and vaccine-protected monkeys at the epitope level.

Algorithms↗

PubMed1992

Evaluation of feline immunodeficiency virus and feline leukemia virus transmembrane peptides for serological diagnosis.

The general model for retrovirus transmembrane (TM) proteins proposed by Gallaher et al. (W. R. Gallaher, J. M. Ball, R. F. Garry, M. C. Griffin, and R. C. Montelaro, AIDS Res. Hum. Retroviruses 5:431-440, 1989) suggests that all retrovirus TM proteins may contain an immunodominant domain (Imd-TM peptide) located at the apex of the TM polypeptide. Although this Imd-TM peptide has been shown to be immunodominant in a variety of lentivirus infections, there has not been a detailed serological analysis of an oncovirus Imd-TM peptide as a diagnostic agent. We describe here an analysis of the antigenic properties and diagnostic potentials of the predicted Imd-TM peptides of feline immunodeficiency virus (FIV) and feline leukemia virus (FeLV) in serological assays of sera from infected cats. The results of these studies demonstrate that antibodies specific to the FIV Imd-TM peptide are detected within 2 weeks postinfection, are maintained at high levels for extended periods, and are not detectable in uninfected or FeLV-infected cats. In marked contrast, the FeLV Imd-TM peptide displayed only negligible levels of serological reactivity in FeLV-infected cats. These studies indicate that the peptide is a useful reagent for the detection of antibodies to FIV.

Amino Acid Sequence↗

PubMed1992

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.

Amino Acid Sequence↗

PubMed1992

Distinct subsets of retroviruses encode dUTPase.

The nonprimate lentiviruses feline immunodeficiency virus, equine infectious anemia virus, visna virus, and caprine encephalitis virus contain a gene segment in the polymerase gene that is lacking in the primate lentiviruses. A related sequence has been noted in other retroviruses, most notably the type D retroviruses. Computer searches have indicated a relatedness between this unique gene segment, termed proteaselike element and elements of both the aspartate proteinase and the dUTPase enzyme families. In this report, we show that members of both nonprimate lentiviruses and type D retroviruses possess dUTPase activity and present a formal demonstration that in feline immunodeficiency virus, the activity is encoded by the proteaselike element.

Amino Acid Sequence↗

PubMed1992

Efficacy of inactivated whole-virus and subunit vaccines in preventing infection and disease caused by equine infectious anemia virus.

We report here on a series of vaccine trials to evaluate the effectiveness of an inactivated equine infectious anemia virus (EIAV) whole-virus vaccine and of a subunit vaccine enriched in EIAV envelope glycoproteins. The inactivated vaccine protected 14 of 15 immunized ponies from infection after challenge with at least 10(5) 50% tissue culture-infective doses of the homologous prototype strain of EIAV. In contrast, it failed to prevent infection in any of 15 immunized ponies that were challenged with the heterologous PV strain. Levels of PV virus replication and the development of disease, however, were significantly reduced in 12 of the 15 ponies so challenged. The subunit vaccine prevented infection from homologous challenge in four of four ponies tested but failed to prevent infection in all four challenged with the PV strain. Two of the four subunit vaccinates had more severe symptoms of equine infectious anemia than nonimmunized ponies infected in parallel. Both vaccines stimulated EIAV-specific cell-mediated immunity. The in vitro lymphoproliferative response was shown to be mediated by T lymphocytes and appeared to be indistinguishable from that induced by EIAV infection. Significant differences were observed in the in vivo lymphocyte responses following challenge with the two virus strains. While peripheral blood mononuclear cells from the inactivated virus vaccinates were equally stimulated by both the prototype and PV strains, the subunit vaccinates challenged with PV exhibited lower levels of spontaneous proliferation and serine esterase activity. This diminished cellular response to PV was correlated with more severe clinical disease in the same ponies. These studies demonstrate for the first time that both an EIAV inactivated whole-virus vaccine and a viral envelope glycoprotein-based subunit vaccine can provide protection against rigorous challenge levels of homologous virus but are unable to protect against similar challenge levels of a heterologous virus. Moreover, the data demonstrate that protection can be achieved in the absence of detectable levels of virus-specific neutralizing antibody in the vaccine recipients at the time of virus challenge. While vaccine-induced virus-specific cell-mediated immune responses were detected, their role in conferring protection was not obvious. Nevertheless, protection from disease appeared to be correlated with the induction of high levels of serine esterase activity following challenge. A significant observation is that while the whole-virus vaccine was usually capable of preventing or markedly moderating disease in the PV-infected ponies, the subunit vaccine appeared to have a high potential to enhance the disease induced by PV infection.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1992

Equine infectious anemia virus gene expression: characterization of the RNA splicing pattern and the protein products encoded by open reading frames S1 and S2.

The utilization of predicted splice donor and acceptor sites in generating equine infectious anemia virus (EIAV) transcripts in fetal donkey dermal cells (FDD) was examined. A single splice donor site identified immediately upstream of the gag coding region joins the viral leader sequence to all downstream exons of spliced EIAV transcripts. The predominant 3.5-kb transcript synthesized in EIAV-infected FDD cells appears to be generated by a single splicing event which links the leader sequence to the first of two functional splice acceptor sites near the 5' end of the S1 open reading frame (ORF). The translation products encoded by the 3.5-kb transcript were examined by producing in vitro transcripts from a cDNA corresponding to this RNA followed by in vitro translation in wheat germ extracts. These transcripts directed the synthesis of three proteins: the virus trans-activator protein (EIAV Tat) encoded by ORF S1, a protein of unknown function encoded by ORF S2, and the virus envelope glycoprotein. When transfected into FDD cells, this cDNA also directed expression of EIAV Tat. Amino-terminal sequence analysis of the in vitro-synthesized S1 protein supports the suggestion that translation of EIAV Tat is initiated at a CUG codon within the virus leader region. Both in vitro-synthesized S2 protein and synthetic peptides corresponding to S2 are shown to react positively with sera obtained from EIAV-infected horses, providing the first direct evidence of expression of this protein in infected animals.

Amino Acid Sequence↗

PubMed1991

Purification and partial characterization of equine infectious anemia virus reverse transcriptase.

Previously we raised a rabbit monospecific antibody (C2003) against a synthetic peptide derived from a sequence within the C-terminal portion of the reverse transcriptase (RT) of the human immunodeficiency virus type 1 (HIV-1). This sequence is found to be conserved in the predicted amino acid sequence of a related lentivirus, the equine infectious anemia virus (EIAV). It was previously determined that the C2003 antibody could cross-react with native EIAV RT and directly inhibit the DNA polymerase activity of the enzyme. We have now fractionated EIAV RT by immunoaffinity chromatography with immobilized C2003 antibody. The procedure yielded an equimolar mixture of two proteins of 66 and 51 kDa associated with both DNA polymerase and RNase H activities. When the EIAV RT proteins were examined by in situ activity gel assays, polymerase activity was found to be principally associated with the 66-kDa component. The fidelity of DNA synthesis by EIAV RT was found to be equivalent to that of HIV-1 RT and lower than that of AMV RT. These observations indicate that the RTs of EIAV and HIV-1 share similar structural and functional properties.

Amino Acid Sequence↗

PubMed1991

Transient suppression of equine immune responses by equine infectious anemia virus (EIAV).

Suppression of the immune system is a common aspect of the disease pathogenesis associated with retroviral infections in both man and animals. We have measured transient suppression of the equine immune system as a loss or decrease in antigen-specific and polyclonal lymphocyte proliferation following experimental infection of ponies with three variants of equine infectious anemia virus (EIAV) with difference virulence characteristics. The transient suppression of proliferative responses was temporally associated with recurrent febrile episodes, which are the hallmark symptom of EIAV-induced disease. Decreased proliferative responses occurred at all times when EIAV viremia was identified, based on the detection of an infectious virus in plasma or viral proteins on peripheral blood mononuclear cells. The immunosuppression was observed most frequently in ponies infected with virulent variants of EIAV which suggested that this effect may contribute to disease pathogenesis. Suppression of polyclonal proliferative responses was induced in vitro by the addition of either infectious or heat-inactivated EIAV to cultures, demonstrating that the viral structural proteins were immunosuppressive in the absence of infection. These studies indicated that EIAV is similar to other retroviruses in that it has the ability to suppress the immune system.

Animals↗

PubMed1991

A structural correlation between lentivirus transmembrane proteins and natural cytolytic peptides.

Although oncoviruses and lentiviruses replicate by similar mechanisms, they differ fundamentally in the usual fate of the infected host cell during productive natural infections. Oncoviruses typically establish persistent nonlytic infections in natural host cells, while lentivirus infections characteristically result in a variety of cytopathic effects ultimately leading to death of the target cell. Described here is a unique structural motif consisting of a strongly amphipathic and arginine-rich helical peptide segment in the carboxyl end of lentivirus TM proteins that is structurally similar to the family of cytolytic peptides produced as defensive agents by certain insects and amphibians. Also demonstrated is the lytic nature of synthetic peptides constructed from the transmembrane (TM) protein of human and simian immunodeficiency viruses (HIV and SIV). Thus, it appears that the cytopathic properties of lentiviruses may be in part attributed to the presence of lytic peptides within the TM protein, designated lentivirus lytic peptide (LLP) and that variations in this segment could account for some of the differences observed in the cytopathicity among variants of a particular lentivirus.

Amino Acid Sequence↗

PubMed1991

Efficacy of SIV/deltaB670 glycoprotein-enriched and glycoprotein-depleted subunit vaccines in protecting against infection and disease in rhesus monkeys.

Immunization with an inactivated whole-virus vaccine is highly effective in preventing lentivirus infection. The viral protein(s) essential to the induction of protective responses, however, have not been identified. To define the role of virion components in the induction of protective immunity, we evaluated the efficacy of glycoprotein-enriched and glycoprotein-depleted simian immunodeficiency virus (SIV) subunit vaccines prepared by lentil-lectin affinity chromatography of gradient-purified virions using the immunization and challenge regimen previously found successful with an inactivated whole-virus vaccine. Infection was determined by successful recovery of virus, the induction of SIV-specific antibody responses, and infection of naive recipients by inoculation with lymph-node-derived lymphocytes from the vaccinates. Immunization with the glycoprotein-enriched preparation prevented infection in two out of four monkeys, whereas the glycoprotein-depleted vaccine failed to prevent infection in all four vaccinates tested. However, the glycoprotein-depleted vaccine appeared to moderate the progression of SIV-induced disease compared with non-immunized infected control monkeys inoculated with the same challenge dose. These data suggest that subunit vaccines containing sufficient quantities of viral glycoproteins can protect against SIV infection, whereas subunit vaccines composed predominantly of viral core proteins cannot. The development of effective vaccines against HIV infection should include studies on the optimum presentation of the viral envelope glycoproteins to produce long-term broadly protective immune responses.

Animals↗

PubMed1991

Characterization of the antigenic domains of the major core protein (p26) of equine infectious anemia virus.

A panel of recombinant trpLE-gag fusion proteins and synthetic peptides was used in Western immunoblot and enzyme-linked immunosorbent assays to identify segments of the major core protein (p26) of equine infectious anemia virus that are antigenic in horses during experimental and natural infections with the virus. The predominant humoral immune response was directed toward a highly immunogenic domain composed of 83 amino acids from the carboxy terminus of p26. The observed immunogenicity of p26 resembled that reported for p24 of human immunodeficiency virus type 1, suggesting the conservation of structural motifs in the lentiviral core proteins which are responsible for their observed immunogenicity during persistent lentivirus infections.

Animals↗

PubMed1991

Analysis of equine humoral immune responses to the transmembrane envelope glycoprotein (gp45) of equine infectious anemia virus.

Defined segments of the transmembrane envelope glycoprotein (gp45) of equine infectious anemia virus were expressed as TrpLE fusion proteins and examined for their reactivity in Western immunoblots against a diverse panel of equine immune sera. The most immunogenic region of gp45 was localized to its amino terminus, positioned between the hydrophobic fusion and the transmembrane domains. A series of overlapping synthetic peptides were used in enzyme-linked immunosorbent assays to define an immunodominant epitope within this region. In contrast, the carboxy-terminal half of gp45 displayed both weak and variable immunoreactivity with equine immune sera.

Amino Acid Sequence↗

PubMed1990

Analysis of antibody reactivities in ELISA using protein blots as antigen substrates: S-ELISA.

We describe here a novel immunoassay procedure, designated strip-ELISA (S-ELISA), in which specific antigens are purified by SDS-PAGE, transferred to support membranes, and utilized in situ as substrate in routine ELISA procedures. Using two different lentivirus systems, simian immunodeficiency virus and equine infectious anemia virus, we demonstrate the utility of S-ELISA for screening hybridoma supernatants during production of monoclonal antibodies and for the dissection of polyclonal antibody responses into defined antigen specificities. The data presented here indicates that the S-ELISA combines the sensitivity of conventional ELISA with the specificity afforded by Western blotting procedures, producing a relatively simple and versatile assay for evaluating antibody reactivities.

Animals↗

PubMed1990

Equine infectious anemia virus (EIAV) humoral responses of recipient ponies and antigenic variation during persistent infection.

Three ponies were inoculated with plasma containing 10(4.8) TCID50 of equine infectious anemia virus (EIAV) and observed for 165 to 440 days. Each pony developed a febrile response within 3 weeks of infection during which a plasma viremia greater than or equal to 10(3.5) TCID50/ml was observed. Analyses of four isolates from sequential febrile episodes in a single pony were conducted by two-dimensional tryptic peptide maps and with monoclonal antibodies in immunoblots. Structural and antigenic alterations were observed in the envelope glycoproteins gp90 and gp45, with greatest variation in gp90. Specific IgG to EIAV gp90, gp45, and p26 of homologous and heterologous isolates was detectable by immunoblots within one month after infection although IgG levels to gp45 at this time were relatively low. The group-specific determinants of gp90 and gp45 were more antigenic than those of p26; however, binding of IgG to these determinants did not correlate with neutralization of EIAV as assayed in fetal equine kidney cells. Neutralizing antibodies were first detectable within two months of infection and only neutralized viruses isolated prior to serum collection. Neutralizing activity of sera collected later in the infection was broadly reactive regardless of the number of clinical episodes the donor had suffered.

Animals↗

PubMed1990

In vitro isolation of a neutralization escape mutant of equine infectious anemia virus (EIAV).

A neutralization escape mutant (A/1 E) of equine infectious anemia virus was isolated after 13 passages in cell culture in the presence of serum containing antibodies to type- and group-specific determinants of EIAV envelope glycoproteins. Loss of neutralization by the selecting serum correlated with loss of two epitopes in the major envelope glycoprotein gp90 of A/1 E which were present in a parallel variant isolated from a persistently infected pony.

Animals↗

PubMed1990

Cryopreservation of equine mononuclear cells for immunological studies.

A rapid and simple technique for the cryopreservation and recovery of equine mononuclear cells was developed. Buffy-coat leukocytes were frozen in autologous plasma containing 10% DMSO and mononuclear cells were recovered by gradient sedimentation using a standard Ficoll-Hypaque purification procedure. The total numbers of mononuclear cells recovered from cryopreserved samples were 94%-82% of those recovered from fresh blood samples. The functional capabilities of the mononuclear cells from cryopreserved buffy coat preparations were compared with those of mononuclear cells from fresh samples by measuring the ability of cells to proliferate in response to mitogens and specific antigens. Cell-surface antigen expression was measured using monoclonal antibodies in conjunction with flow cytometric techniques and alloantisera in a complement mediated cytotoxicity assay. Cryopreserved mononuclear cells were capable of proliferating normally when stimulated with several mitogens, pokeweed mitogen, phytohemagglutinin and concanavalin A, and a single specific antigen preparation, equine influenza-2 (Equi-2) proteins. The maximum levels of proliferation induced by varying the concentrations of mitogens or the Equi-2 proteins were the same for both the fresh and cryopreserved cells. However, the cryopreserved cells usually required one more day in culture to attain maximum proliferation levels. Flow cytometric analysis of the samples demonstrated that the relative proportions of different lymphocyte populations were not altered by the cryopreservation step. Similarly, MHS alloantigen expression was not altered. The simplicity of the technique coupled with the retained functional properties allows for the cryopreservation of large numbers of leukocytes and the ability to assay various immune functions at a later time.

Animals↗

PubMed1990

Biochemical and immunological characterization of the major structural proteins of feline immunodeficiency virus.

Feline immunodeficiency virus (FIV) structural proteins were identified using sera obtained from experimentally inoculated cats. Proteins analysed by both radioimmunoprecipitation and Western blotting were specific for FIV infection and failed to cross-react with either antisera to feline leukaemia virus of feline syncytium-forming virus. Western blot analysis of purified virus revealed immunoreactive proteins with apparent Mr of 65K, 50K, 40K, 32K, 24K, 15K and 10K. The major core structural proteins of the virus were isolated by reverse phase HPLC and the aminoterminal sequences of p10 and p24 were determined. Monoclonal antibodies specific for p24 suggested the presence of a precursor protein that could be detected in 35[S]methionine/cysteine-labelled, virus-infected cell extracts. This putative precursor protein possessed an apparent Mr of 50K (Pr50gag). Further analysis revealed the presence of two additional proteins of 130K and 40K. Experiments utilizing tunicamycin, endoglycosidase H and glycopeptidase F revealed that p130 and p40 exhibited properties characteristic of glycoproteins. Our studies also indicated that FIV is immunologically related to other lentiviruses.

Amino Acid Sequence↗