Multiple RNA splicing and the presence of cryptic RNA splice donor and acceptor sites may contribute to low expression levels and poor immunogenicity of potential DNA vaccines containing the env gene of equine infectious anemia virus (EIAV).

The env gene is an excellent candidate for inclusion in any DNA-based vaccine approach against equine infectious anemia virus (EIAV). Unfortunately, this gene is subjected to mutational pressure in E. coli resulting in the introduction of stop codons at the 5' terminus unless it is molecularly cloned using very-low-copy-number plasmid vectors. To overcome this problem, a mammalian expression vector was constructed based on the low-copy-number pLG338-30 plasmid. This permitted the production of full-length EIAV env gene clones (plcnCMVenv) from which low-level expression of the viral surface unit glycoprotein (gp90) was detected following transfection into COS-1 cells. Although this suggested the nuclear export of complete env mRNA moieties at least two additional polypeptides of 29 and 20kDa (probably Rev) were produced by alternative splicing events as demonstrated by the fact that their synthesis was prevented by mutational inactivation of EIAV env splice donor 3 (SD3) site. The plcnCMVenv did not stimulate immune responses in mice or in horses, whereas an env construct containing an inactivated SD3 site (plcnCMVDeltaSD3) did induce weak humoral responses against gp90 in mice. This poor immunogenicty in vivo was probably not related to the inherent antigenicity of the proteins encoded by these constructs but to some fundamental properties of EIAV env gene expression. Attempts to modify one of these properties by mutational inactivation of known viral RNA splice sites resulted in activation of previously unidentified cryptic SD and slice acceptor sites.

Derivation and characterization of a live attenuated equine influenza vaccine virus.

OBJECTIVE: To develop and characterize a cold-adapted live attenuated equine-2 influenza virus effective as an intranasal vaccine. ANIMALS: 8 ponies approximately 18 months of age. PROCEDURES: A wild-type equine-2 virus, A/Equine/Kentucky/1/91 (H3N8), was serially passaged in embryonated chicken eggs at temperatures gradually reduced in a stepwise manner from 34 C to 30 C to 28 C to 26 C. At different passages, infected allantoic fluids were tested for the ability of progeny virus to replicate in Madin-Darby canine kidney (MDCK) cells at 34 C and 39.5 C. Virus clones that replicated at 26 C in eggs and at 34 C in MDCK cells, but not at 39.5 C in MDCK cells, were tested for stability of the cold-adapted, temperature-sensitive (ts), and protein synthesis phenotypes. A stable clone, P821, was evaluated for safety, ability to replicate, and immunogenicity after intranasal administration in ponies. RESULTS: Randomly selected clones from the 49th passage were all ts with plaquing efficiencies of < 10(-6) (ratio of 39.5 C:34 C) and retained this phenotype after 5 serial passages at 34 C in either embryonated eggs or MDCK cells. The clone selected as the vaccine candidate (P821) had the desired degree of attenuation. Administered intranasally to seronegative ponies, the virus caused no adverse reactions or overt signs of clinical disease, replicated in the upper portion of the respiratory tract, and induced a strong serum antibody response. CONCLUSION AND CLINICAL RELEVANCE: A candidate live attenuated influenza vaccine virus was derived by cold-adaptation of a wild-type equine-2 influenza virus, A/Equine/Kentucky/1/91, in embryonated eggs.

Safety, efficacy, and immunogenicity of a modified-live equine influenza virus vaccine in ponies after induction of exercise-induced immunosuppression.

OBJECTIVE: To determine safety, efficacy, and immunogenicity of an intranasal cold-adapted modified-live equine influenza virus vaccine administered to ponies following induction of exercise-induced immunosuppression. DESIGN: Prospective study. ANIMALS: Fifteen 9- to 15-month old ponies that had not had influenza. PROCEDURE: Five ponies were vaccinated after 5 days of strenuous exercise on a high-speed treadmill, 5 were vaccinated without undergoing exercise, and 5 were not vaccinated or exercised and served as controls. Three months later, all ponies were challenged by nebulization of homologous equine influenza virus. Clinical and hematologic responses and viral shedding were monitored, and serum and nasal secretions were collected for determination of influenza-virus-specific antibody isotype responses. RESULTS: Exercise caused immunosuppression, as indicated by depression of lymphocyte proliferation in response to pokeweed mitogen. Vaccination did not result in adverse clinical effects, and none of the vaccinated ponies developed clinical signs of infection following challenge exposure. In contrast, challenge exposure caused marked clinical signs of respiratory tract disease in 4 control ponies. Vaccinated and control ponies shed virus after challenge exposure. Antibody responses to vaccination were restricted to serum IgGa and IgGb responses in both vaccination groups. After challenge exposure, ponies in all groups generated serum IgGa and IgGb and nasal IgA responses. Patterns of serum hemagglutination inhibition titers were similar to patterns of IgGa and IgGb responses. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggested that administration of this MLV vaccine to ponies with exercise-induced immunosuppression was safe and that administration of a single dose to ponies provided clinical protection 3 months later.

Transcutaneous immunization of domestic animals: opportunities and challenges.

Transcutaneous immunization (TCI), the topical application of antigen and adjuvant directly onto intact skin, can safely and effectively elicit systemic immune responses in mice and humans against a variety of antigens. This novel method of vaccine delivery has the potential to provide a safe and convenient method by which vaccines may be delivered to elicit protective immunity in domestic animals. To date, however, immune responses induced by TCI in companion and production animals has not been reported. In this report, we demonstrate that TCI may be widely applicable to many animals. Immune responses elicited by TCI require further optimization for each antigen and species, and success may depend upon the structure and composition of the skin of the target species. The prospect of TCI as a practical and broadly applicable approach to vaccination in veterinary medicine is discussed in the context of these challenges.

Tissue sites of persistent infection and active replication of equine infectious anemia virus during acute disease and asymptomatic infection in experimentally infected equids.

Equine infectious anemia virus (EIAV) infection of horses is characterized by recurring cycles of disease and viremia that typically progress to an inapparent infection in which clinical symptoms are absent as host immune responses maintain control of virus replication indefinitely. The dynamics of EIAV viremia and its association with disease cycles have been well characterized, but there has been to date no comprehensive quantitative analyses of the specific tissue sites of EIAV infection and replication in experimentally infected equids during acute disease episodes and during asymptomatic infections in long-term inapparent carriers. To characterize the in vivo site(s) of viral infection and replication, we developed a quantitative competitive PCR assay capable of detecting 10 copies of viral DNA and a quantitative competitive reverse transcription-PCR assay with a sensitivity of about 30 copies of viral singly spliced mRNA. Animals were experimentally infected with one of two reference viruses: the animal-passaged field isolate designated EIAV(Wyo) and the virulent cell-adapted strain designated EIAV(PV). Tissues and blood cells were isolated during the initial acute disease or from asymptomatic animals and analyzed for viral DNA and RNA levels by the respective quantitative assays. The results of these experiments demonstrated that the appearance of clinical symptoms in experimentally infected equids coincided with rapid widespread seeding of viral infection and replication in a variety of tissues. During acute disease, the predominant cellular site of viral infection and replication was the spleen, which typically accounted for over 90% of the cellular viral burden. In asymptomatic animals, viral DNA and RNA persisted in virtually all tissues tested, but at extremely low levels, a finding indicative of tight but incomplete immune control of EIAV replication. During all disease states, peripheral blood mononuclear cells (PBMC) were found to harbor less than 1% of the cellular viral burden. These quantitative studies demonstrate that tissues, rather than PBMC, constitute the predominant sites of virus replication during acute disease in infected equids and serve as resilient reservoirs of virus infection, even in the presence of highly effective immune responses that maintain a stringent control of virus replication in long-term inapparent carriers. Thus, these observations with EIAV, a predominantly macrophage-tropic lentivirus, highlight the role of tissues in sequestering lentiviral infections from host immune surveillance.

Effects of long terminal repeat sequence variation on equine infectious anemia virus replication in vitro and in vivo.

The long terminal repeat (LTR) is reported to be one of the most variable portions of the equine infectious anemia virus (EIAV) genome. To date, however, no information is available on the effects of observed sequence variations on viral replication properties, despite a widespread assumption of the biological importance of EIAV LTR variation. EIAV LTR sequence variability is confined mostly to a small portion of the enhancer within the U3 segment of the LTR. Analysis of published EIAV LTR sequences revealed six different types of LTR based on the pattern of putative transcription factor motifs within the variable region of the enhancer. To test directly the significance of LTR variation, the in vitro and in vivo replication properties of two variant LTR species were investigated using two isogenic viruses, EIAV(19-2) and EIAV(19-2-6A), differing only within the enhancer region. The results of these studies demonstrated that the two variants replicated with similar kinetics and to equal levels in cultured equine fibroblasts or in equine macrophage, the natural target cell of EIAV, even after prolonged serial passage in the latter cell type. Furthermore, EIAV(19-2) and EIAV(19-2-6A) variants demonstrated similar replication levels in experimentally infected ponies. However, ponies infected with EIAV(19-2-6A) exhibited a rapid switch in the prevalent LTR type, such that by 112 days postinfection, no original-LTR-type viruses were evident. This specific and rapid shift in LTR quasispecies indicates an in vivo selection that is not reflected in simple in vitro replication rates, suggesting undefined selection pressures in vivo that drive LTR variation during persistent EIAV infection.

Immunization of dogs and cats with a DNA vaccine against rabies virus.

The applicability of DNA immunization technology for vaccine development in companion animals was investigated by immunizing dogs and cats by the intramuscular (i.m.) and intradermal (i.d.) routes with a plasmid DNA vector encoding the rabies virus glycoprotein G. In dogs, administration of 100 microg DNA doses by the i.m. route resulted in stronger and more durable rabies virus neutralizing antibody (RVNA) titers than those obtained by i.d. inoculation. In contrast, i.m. vaccination of cats with a similar dose was less effective in terms of mean titer and seroconversion frequency. However, efficacy was improved by increasing the dosage to 300 microg of DNA per immunization. Interestingly, i.d. inoculation of cats appeared to be a superior route of delivery in this species, resulting in higher seroconversion frequency than i.m. administration. In addition, geometric mean RVNA titers in i.d. inoculated cats increased over four-fold during a seven month period following a second and final immunization. These results demonstrate that non-facilitated, naked DNA vaccines can elicit strong, antigen-specific immune responses in dogs and cats, and DNA immunization may be a useful tool for future development of novel vaccines for these species.

Cloning of a family of serine protease genes from the cat flea Ctenocephalides felis.

Serine protease gene fragments approximately 480 nucleotides in length were amplified from Ctenocephalides felis larval and adult cDNA libraries using degenerate oligonucleotide PCR primers. Partial clones of thirty-eight distinct serine protease encoding sequences were isolated, and nineteen different full-length cDNAs encoding mature serine proteases were subsequently cloned and sequenced. All of the mature proteases contained the histidine, aspartic acid and serine amino acids of the catalytic triad characteristic of serine proteases. The mature C. felis serine proteases had amino acid sequences that were at most 29-53% identical to those known insect and arachnid serine proteases. Two of the C. felis gene sequences had similarity with the Drosophila melanogaster developmental genes snake and stubble. mRNA expression of selected serine protease genes was examined in different life stages, tissues, genders, and in response to bloodfeeding.

Detection of equine infectious anemia viral RNA in plasma samples from recently infected and long-term inapparent carrier animals by PCR.

Control of equine infectious anemia (EIA) is currently based on detection of anti-EIA virus (EIAV) antibodies. However, serologic diagnostic methods may give false-negative results in infected horses that fail to respond adequately or are in the early stages of infection. We developed a reverse transcriptase nested PCR (RT-nPCR) assay for the detection of viral gag gene sequences in plasma from EIAV-infected horses. The ability of RT-nPCR to detect field strains of EIAV was investigated by assaying plasma samples from 71 horses stabled on EIA quarantine ranches. Positive PCR signals were detected in 63 of 63 horses with EIAV antibody test-positive histories on approved serologic tests, demonstrating that RT-nPCR was probably directed against highly conserved sequences in the viral genome. The RT-nPCR assay, agar gel immunodiffusion test, and conventional virus isolation were compared for detection of early infection in 12 experimentally infected ponies. Viral gag sequences were detected in all 12 animals by 3 days postinfection (p.i.) by RT-nPCR, whereas virus could not be routinely isolated on cell culture until 9 to 13 days p.i. and EIAV antibodies could not be detected by agar gel immunodiffusion until 20 to 23 days p.i. Finally, specificity of the RT-nPCR assay was examined by testing plasma from 43 horses with serologic test-negative histories and no known contact with EIAV-infected animals. Viral gag sequences were not detectable in this control group. These data suggest that the EIAV RT-nPCR assay effectively detects EIAV and is more sensitive than current standard methods for detection of early stages of infection.

Replication in vitro and in vivo of an equine infectious anemia virus mutant deficient in dUTPase activity.

As an important enzyme in DNA synthesis, dUTPase is present in a wide variety of organisms and viruses and has been identified as a component of the equine infectious anemia virus (EIAV) pol gene. The role of EIAV dUTPase, designated DU, in virus replication in vitro and in vivo was investigated with a recently described infectious molecular clone of EIAV. A deletion mutant that was deficient in dUTPase activity was constructed, and its replication kinetics was examined in fetal equine kidney (FEK) cells and primary equine bone marrow macrophage (EBMM) cells. In FEK cells, which are permissive for EIAV replication, the mutant virus replicated as well as the parental virus. In primary cultures of EBMM cells, which are primary targets of EIAV infection in vivo, the DU mutant showed delayed replication kinetics and replicated to a lower extent than did the parental virus. As the multiplicity of infection decreased, the difference between the parental and mutant viruses increased, such that at the lowest multiplicity of infection tested, there was over a 100-fold difference in virus production. The mutant virus was also much less cytopathic. The role of DU in replication in vivo was examined using a Shetland pony model of EIAV infection. Shetland ponies that were infected with the parental and mutant viruses showed transient virus RNA levels in plasma approximately 5 to 10 days postinfection. The peak virus levels in plasma (as measured by a quantitative reverse transcriptase PCR assay) were 10- to 100-fold lower in the mutant virus-infected animals than in the animals infected with the parental virus. However, ponies infected with the mutant virus mounted similar antibody responses despite the marked differences in virus replication. These studies demonstrate that EIAV DU is important for the efficient replication of the virus in macrophages in vitro and in vivo and suggests that variations in the DU sequence could markedly affect the biological and pathogenic properties of EIAV.

Enhanced sensitivity to neutralizing antibodies in a variant of equine infectious anemia virus is linked to amino acid substitutions in the surface unit envelope glycoprotein.

Serial passage of the prototype (PR) cell-adapted Wyoming strain of equine infectious anemia virus (EIAV) in fetal donkey dermal (FDD) rather than fetal horse (designated fetal equine kidney [FEK]) cell cultures resulted in the generation of a variant virus strain which produced accelerated cytopathic effects in FDD cells and was 100- to 1,000-fold more sensitive to neutralizing antibodies than its parent. This neutralization-sensitive variant was designated the FDD strain. Although there were differences in glycosylation between the PR and FDD strains, passage of the FDD virus in FEK cells did not reduce its sensitivity to neutralizing antibody. Nucleotide sequencing of the region encoding the surface unit (SU) protein from the FDD strain revealed nine amino acid substitutions compared with the PR strain. Two of these substitutions resulted in changes in the polarity of charge, four caused the introduction of a charged residue, and three had no net change in charge. Nucleotide sequence analysis was extended to the region of the FDD virus genome encoding the extracellular domain of the transmembrane envelope glycoprotein (TM). Unlike the situation with the FDD virus coding region, there were minor variations in nucleotide sequence between individual molecular clones containing this region of the TM gene. Although each clone contained three nucleotide substitutions compared with the PR strain, only one of these was common to all, and this did not affect the amino acid content. Of the remaining two nucleotide substitutions, only one resulted in an amino acid change, and in each case, this change appeared to be conservative. To determine if amino acid substitutions in the SU protein of FDD cell-grown viruses were responsible for the enhanced sensitivity to neutralizing antibodies, chimeric viruses were constructed by using an infectious molecular clone of EIAV. These chimeric viruses contained all of the amino acid substitutions found in the FDD virus strain and were significantly more sensitive to neutralizing antibodies than viruses from the parental (PR) molecular clone. These results demonstrated that sensitivity to neutralizing antibodies in EIAV can be conferred by amino acid residues in the SU protein. However, such amino acid substitutions were not sufficient to enhance cytopathogenicity, as the chimeric viruses did not cause excessive degenererative effects in FDD cells, as was observed with the parental FDD virus strain.

Lentivirus cross-reactive determinants present in the capsid protein of equine infectious anaemia virus.

In this study we used immune sera from equine infectious anaemia virus (EIAV)-infected horses which uniquely display broad reactivity with different lentivirus capsid proteins (CA) to characterize the cross-reactive determinants of lentivirus CA proteins. In particular, the role of the major homology region (MHR) of lentivirus CA proteins in this serological cross-reactivity was evaluated using both equine immune serum and murine monoclonal antibodies (MAbs) directed against the MHR segment of different lentiviruses. The results of our studies indicate that about 80% of sera from long-term experimentally infected ponies or naturally infected horses react with human immunodeficiency virus type 1 CA in Western immunoblot assays. In addition, the cross-reactive determinants on the EIAV CA were localized within the immunodominant carboxyl terminus of the protein (residues 277 to 367). However, the cross-reactive determinants recognized by the equine sera do not appear to correlate with linear peptides from the carboxyl terminus of the EIAV CA, including the MHR. These results suggest cross-reactivity between more distant lentiviruses is associated with non-linear determinants. In contrast, MHR-specific MAbs did react with linear peptides by ELISA and distinguished the primate lentiviruses from EIAV and feline immunodeficiency virus. These data support the concept of a highly conserved structural and antigenic organization among the CA proteins of lentiviruses from different species.

Enhancement of EIAV replication and disease by immunization with a baculovirus-expressed recombinant envelope surface glycoprotein.

The potential for antibody-dependent enhancement of replication of macrophage/monocyte tropic viruses has posed a significant problem in the development of vaccines for several animal and human viruses and has raised significant concern in the design of potential AIDS vaccines. Using the previously described equine infectious anemia virus/Shetland pony system as a model for HIV-1 vaccine development, we have evaluated the efficacy of a recombinant subunit vaccine containing a baculovirus-expressed envelope surface glycoprotein (gp90) of EIAV. The results of these trials demonstrate not only that the recombinant vaccine failed to protect against infection by standard homologous and heterologous EIAV challenge strains, but that it resulted in a marked enhancement of virus replication and exacerbation of disease in immunized ponies exposed to the heterologous virus strain. Thus, the recombinant EIAV gp90 vaccine provides a novel in vivo model for examining in detail the mechanisms of immune enhancement of a lentivirus infection and for evaluating strategies to avoid the production of deleterious immune responses in AIDS vaccine design.

Characterization of infectious molecular clones of equine infectious anaemia virus.

We have recovered five infectious molecular clones of the lentivirus equine infectious anaemia virus (EIAV). The clones were recovered from fetal equine kidney (FEK) cells infected with a virulent, cell culture-adapted virus stock (designated PV) and have been characterized at a molecular level. Each clone has unique envelope and long terminal repeat (LTR) sequences. We further investigated LTR sequence variation in the PV stock using PCR amplification to obtain additional LTR clones from infected FEK cells and from peripheral blood mononuclear cells (PBMCs) from animals experimentally infected with PV. Sequence analysis of resulting clones indicates a selection for different LTR populations in pony PBMCs compared to FEK cells. Finally, we observed that the cloned EIAV proviruses did not remain infectious when maintained in a derivative of pBR322. However, two proviruses have been stably maintained in a low copy number vector (pLG338-SPORT).

Characterization of equine infectious anemia virus dUTPase: growth properties of a dUTPase-deficient mutant.

The putative dUTPase domain was deleted from the polymerase (pol) gene of equine infectious anemia virus (EIAV) to produce a recombinant delta DUpol Escherichia coli expression cassette and a delta DU proviral clone. Expression of the recombinant delta DUpol polyprotein yielded a properly processed and enzymatically active reverse transcriptase, as determined by immunoblot analysis and DNA polymerase activity gels. Transfection of delta DU provirus into feline (FEA) cells resulted in production of virus that replicated to wild-type levels in both FEA cells and fetal equine kidney cells. In contrast, the delta DU virus replicated poorly (less than 1% of wild-type levels) in primary equine macrophage cultures, as measured by reverse transcriptase assays. Preparations of delta DU virus contained negligible dUTPase activity, which confirms that virion-associated dUTPase is encoded in the pol gene region between the RNase H domain and integrase, as has been demonstrated previously for feline immunodeficiency virus (J. H. Elder, D. L. Lerner, C. S. Hasselkus-Light, D. J. Fontenot, E. Hunter, P. A. Luciw, R. C. Montelaro, and T. R. Phillips, J. Virol. 66:1791-1794, 1992). Our results suggest that virus-encoded dUTPase is dispensable for virus replication in dividing cells in vitro but may be required for efficient replication of EIAV in nondividing equine macrophages, the natural host cells for this virus.

Lentivirus envelope sequences and proviral genomes are stabilized in Escherichia coli when cloned in low-copy-number plasmid vectors.

A promoter-selection vector (pKK232-8) was used to identify sequences with strong Escherichia coli promoter activity positioned near the start of the envelope-encoding genes (env) of two lentiviruses, simian immunodeficiency virus (SIV) and equine infectious anemia virus (EIAV). For EIAV, cloning the cryptic promoter sequences together with downstream sequences encoding the envelope glycoprotein (gp90) in moderate- to high-copy-number (hcn) plasmid vectors, such as pBR322 or pUC, resulted in rearrangements and point mutations in env when propagated in E. coli. To alleviate this problem, low-copy-number (lcn) cloning vectors, pLG338-30 and pLG339-SPORT, were constructed. The plasmids carry resistance markers for ampicillin (ApR) or kanamycin (KmR), the pSC101 origin of replication (ori) from plasmid pLG338 [Stoker et al., Gene 18 (1982) 335-341], and a multiple cloning site (MCS) from plasmids pIBI30 or pSPORT. Full-length env and genomic proviral sequences of EIAV were genetically stable when subcloned into these lcn vectors. Proviral sequences of an SIV clone (pBK28-SIV) that are genetically unstable in the hcn vector pUC18 were also stabilized and remained fully infectious when subcloned into the lcn vector pLG339-SPORT. These lcn vectors appear to be generally useful in stabilizing lentivirus genomic sequences for subcloning, propagation, and manipulation in E. coli, possibly as a result of reducing the level of toxic gene expression from cryptic promoter sequences.

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.

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.

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.

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.