Modelling Mutation in Equine Infectious Anemia Virus Infection Suggests a Path to Viral Clearance with Repeated Vaccination.

Equine infectious anemia virus (EIAV) is a lentivirus similar to HIV that infects horses. Clinical and experimental studies demonstrating immune control of EIAV infection hold promise for efforts to produce an HIV vaccine. Antibody infusions have been shown to block both wild-type and mutant virus infection, but the mutant sometimes escapes. Using these data, we develop a mathematical model that describes the interactions between antibodies and both wild-type and mutant virus populations, in the context of continual virus mutation. The aim of this work is to determine whether repeated vaccinations through antibody infusions can reduce both the wild-type and mutant strains of the virus below one viral particle, and if so, to examine the vaccination period and number of infusions that ensure eradication. The antibody infusions are modelled using impulsive differential equations, a technique that offers insight into repeated vaccination by approximating the time-to-peak by an instantaneous change. We use impulsive theory to determine the maximal vaccination intervals that would be required to reduce the wild-type and mutant virus levels below one particle per horse. We show that seven boosts of the antibody vaccine are sufficient to eradicate both the wild-type and the mutant strains. In the case of a mutant virus infection that is given infusions of antibodies targeting wild-type virus (i.e., simulation of a heterologous infection), seven infusions were likewise sufficient to eradicate infection, based upon the data set. However, if the period between infusions was sufficiently increased, both the wild-type and mutant virus would eventually persist in the form of a periodic orbit. These results suggest a route forward to design antibody-based vaccine strategies to control viruses subject to mutant escape.

Validation of an immunoblot assay employing an objective reading system and used as a confirmatory test in equine infectious anaemia surveillance programs.

Equine infectious anaemia (EIA) is a blood borne disease that is listed among the notifiable diseases of the World Organisation for Animal Health (OIE). EIA is also regulated by the OIE for the international trading provisions and is generally subject to control programmes. Since 2011, Italy has been conducting a surveillance plan based on a three-tier diagnostic system, using a serological ELISA as screening test, an agar gel immunodiffusion test (AGIDT) as a confirmatory method, and an immunoblot (IB) as an alternative confirmatory assay for discordant results between the first two tests. As for the in-house competitive ELISA (c-ELISA) and the AGIDT, the Italian National Reference Laboratory for EIA (NRL) validated the IB according to the OIE guidelines, employing eight panels containing positive sera, including those from EIA virus (EIAV) proven infected horses, and negative horse, mule and donkey sera collected from different geographical areas. In addition, two international reference image panels were employed for the optimization and the validation of the digital image reading system adopted that allows an impartial measurement of the serum reactivity in the IB assay. The immunological reactivity to EIAV antigens, p26, gp45 and gp90 adsorbed on the IB membrane, determines the serological status of the animal and for EIA, a p26 positive band together with at least one of the other antigen defines a subject as serologically positive for EIAV. For validation, the parameters assessed were threshold values, analytical and diagnostic sensitivity and specificity, repeatability and reproducibility. These parameters were evaluated for each antigen as well as in combination, according to the diagnostic algorithm established above. The validation data defined the IB as having a satisfactory sensitivity, specificity, repeatability and reproducibility for all antigens and species tested. An instrumental recording of the results improves the confidence in using IB as a confirmatory test for EIAV, differently from the AGIDT that is read by an operator. The advantages of using the IB are its higher sensitivity, to that of the AGIDT, which allows an earlier detection of infection that reduces the risk of transmission and therefore the incidence of the EIA, and its higher specificity to that of the ELISA which is based on the discrimination of subjects reacting only against the p26, the antigen used by all ELISAs available, which are not considered as infected by EIAV. In particular, when this assay is used in outbreaks it can detect new cases earlier than the AGIDT, and therefore reduce the restriction period with an economic benefit for the animal owners and the public veterinary sanitary system.

Prospects in Innate Immune Responses as Potential Control Strategies against Non-Primate Lentiviruses.

Lentiviruses are infectious agents of a number of animal species, including sheep, goats, horses, monkeys, cows, and cats, in addition to humans. As in the human case, the host immune response fails to control the establishment of chronic persistent infection that finally leads to a specific disease development. Despite intensive research on the development of lentivirus vaccines, it is still not clear which immune responses can protect against infection. Viral mutations resulting in escape from T-cell or antibody-mediated responses are the basis of the immune failure to control the infection. The innate immune response provides the first line of defense against viral infections in an antigen-independent manner. Antiviral innate responses are conducted by dendritic cells, macrophages, and natural killer cells, often targeted by lentiviruses, and intrinsic antiviral mechanisms exerted by all cells. Intrinsic responses depend on the recognition of the viral pathogen-associated molecular patterns (PAMPs) by pathogen recognition receptors (PRRs), and the signaling cascades leading to an antiviral state by inducing the expression of antiviral proteins, including restriction factors. This review describes the latest advances on innate immunity related to the infection by animal lentiviruses, centered on small ruminant lentiviruses (SRLV), equine infectious anemia virus (EIAV), and feline (FIV) and bovine immunodeficiency viruses (BIV), specifically focusing on the antiviral role of the major restriction factors described thus far.

Enzyme-linked immunosorbent assay and agar gel immunodiffusion assay for diagnosis of equine infectious anemia employing p26 protein fused to the maltose-binding protein.

A codon-optimized equine infectious anemia virus p26 gene was fused to a maltose-binding protein (MBP) and expressed in Escherichia coli for use as an antigen in agar gel immunodiffusion (AGID) and enzyme-linked immunosorbent assay (ELISA) for diagnosis of equine infectious anemia. An analysis of analytical sensitivity and specificity showed that the antigen MBP-p26rec reacted positively with a reference World Organization for Animal Health serum and demonstrated no cross-reaction against sera from vaccinated animals in either test. The diagnostic characteristics were evaluated and presented excellent values. The AGIDrec showed 100% sensitivity and specificity, and the ELISArec showed 100% sensitivity and 99.64% specificity. In addition, MBP-p26rec was stabile after three years of storage at 4 °C, maintaining its immunoreactivity.

Comparison of two agar gel immunodiffusion protocols for diagnosing equine infectious anemia.

This study compared agar gel immunodiffusion (AGID) protocols for diagnosing equine infectious anemia. Two commercial testing kits were used: one following the Japanese Act on Domestic Animal Infectious Diseases Control and one following the World Organisation for Animal Health (OIE) manual. From 651 samples tested, both protocols gave identical results for 647 samples (23 samples tested positive; 624 tested negative). Non-specific reactions were observed in 21 samples testing negative by the Japanese protocol, but none were observed with the OIE protocol. The kappa coefficient value was 0.962, indicating almost perfect agreement between the two protocols. This study found no difference in diagnostic agreement between the two protocols, but the OIE protocol produced non-specific reactions less frequently than the Japanese protocol.

Molecular characterization of equine infectious anaemia virus from a major outbreak in southeastern France.

In 2009, a major outbreak of equine infectious anaemia (EIA) was reported in the south-east of France. This outbreak affected three premises located in the Var region where the index case, a 10-year-old mare that exhibited clinical signs consistent with EIA, occurred at a riding school. Overall, more than 250 horses were tested for EIAV (equine infectious anaemia virus) antibodies, using agar gel immunodiffusion test, and 16 horses were positive in three different holdings. Epidemiological survey confirmed that the three premises were related through the purchase/sale of horses and the use of shared or nearby pastures. Molecular characterization of viruses was performed by sequencing the full gag gene sequence (1,400 bp) of the proviral DNAs retrieved from the spleen of infected animals collected post-mortem. Phylogenetic analysis confirmed epidemiological data from the field, as viruses isolated from the three premises were clustering together suggesting a common origin whereas some premises were 50 km apart. Moreover, viruses characterized during this outbreak are different from European strains described so far, underlying the high genetic diversity of EIAV in Europe.

Dynamics of lentiviral infection in vivo in the absence of adaptive immune responses.

Understanding the dynamics of acute viral infection is crucial for developing strategies to prevent and control infection. In this study, lentiviral dynamics in a host without adaptive immunity were examined in order to determine kinetic parameters of infection and quantify the effect of neutralizing antibodies in preventing infection, using mathematical modeling of data from equine infectious anemia virus (EIAV) infection of horses with severe combined immunodeficiency (SCID). Estimated parameters were used to calculate the basic reproductive number and virus doubling time and found that the rate that antibodies neutralized virus was ~18 times greater than the virus clearance rate. These results establish EIAV replication kinetics in SCID horses and the minimal efficacy of antibodies that blocked infection. Furthermore, they indicate that EIAV is at most mildly cytopathic. This study advances our understanding of EIAV infection and may have important implications for the control of other viral infections, including HIV.

Validation of an indirect ELISA employing a chimeric recombinant gag and env peptide for the serological diagnosis of equine infectious anemia.

The National Reference Center for equine infectious anemia (EIA) validated a commercial ELISA (Eradikit® EIAV Indirect ELISA, In3diagnostic®, Turin, Italy) employing a chimeric recombinant gag and env peptide for the detection of EIA virus antibodies, following the guidelines of the World Organization for Animal Health. The validation parameters evaluated were: analytical sensitivity (Se) and specificity (Sp); diagnostic Se and Sp; precision, based on repeatability and reproducibility through the estimation of the standard deviation (SD) and the coefficient of variation (CV); accuracy, estimated from a multiple K and relative Sp and Se with respect to those of the agar gel immunodiffusion test (AGIDT). Positive and negative predictive values were also defined. The assay showed a high specificity and a limit of detection of 1.43 log10 major than AGIDT. Diagnostic Se was 100% and Sp was 99.3%, while SD values ranged from 1.58 to 5.01 with a CV between 2.8% and 28.8%. Multiple K was 0.98 and relative Se and Sp were respectively 99.1% and 100%. The assay proved to be robust and to possess a high sensitivity in detecting first antibodies produced at onset of infection as well as high analytic and diagnostic Se and Sp values, confirming it as a serological assay fit for purpose within EIA surveillance programs.

Evaluation of six serological ELISA kits available in Italy as screening tests for equine infectious anaemia surveillance.

BACKGROUND: ELISAs are known to have a higher diagnostic sensitivity than the agar gel immunodiffusion (AGID) when employed for serological diagnosis of equine infectious anaemia (EIA). For this purpose, an "in-house" and five commercial ELISAs available in Italy were assessed by the National Reference Centre for EIA for their analytic specificity (Sp); precocity, defined as capability of detecting first antibodies produced during a new infection; precision based on repeatability and reproducibility, estimated from the coefficient of variation (CV); accuracy, estimated from multiple K and relative Sp and sensitivity (Se). Two serum panels, positive for non-equine retroviruses and the most frequent equine viruses, were employed to measure analytic Sp. ELISA precocity was also compared to that of one "in-house" and three commercial AGID kits, employing a panel of sera, collected weekly from horses infected with modified EIA viruses. Precision and accuracy were defined using results of a panel containing positive and negative sera examined in an inter-laboratory trial with the participation of the ten Official Laboratories. Furthermore, a questionnaire was used to assess the appropriateness of each kit for routine use. RESULTS: Analytic Sp was 100%, while the 75th percentile of CVs for positive sera varied from 0.4% to 12.73% for repeatability and from 1.6% to 44.87% for reproducibility. Although CV of the negative serum was constantly high, its outcome was unaltered. Relative Se ranged from 98.2% to 100%, relative Sp was constantly 100% and multiple K ranged from 0.95 to 1. Precocity differed among the assays: three kits detected 4.8% and 42.9% positive samples on 21 days post infection (dpi), all assays detected positive samples on 28 dpi, between 47.6% and 95.2%. Precocity of ELISAs was superior to that of the AGIDs except for two assays. In view of the feedback obtained from the questionnaires, all kits were considered appropriate for routine use. CONCLUSION: All ELISAs having high Se and precocity are preferable as a screening test in EIA surveillance programmes to the AGID tests examined. These two tests can be incorporated in a serial diagnostic pathway to improve the efficacy of a surveillance plan.

A pilot study on interaction between donkey tetherin and EIAV stains with different virulent and replication characteristics.

Tetherin (BST-2) is an important host restriction factor that can inhibit the release of a diverse array of enveloped viruses from infected cells. Conversely, to facilitate their release and spread, many viruses have evolved various strategies to overcome the antiviral effect of tetherin in a species-specific manner. During the development of an attenuated equine infectious anemia virus (EIAV) vaccine in our laboratory, we found that serial passage of a field-isolated virulent EIAV strains in horse and donkey as well as the cultivated donkey cells, produces several typical EIAV strains, including EIAVDV, EIAVDLV, and EIAVFDDV, which exhibit distinct virulence and replication features in vivo and in vitro. However, the role of host restriction factors in EIAV evolution during the serial passage is not well understood. This study aimed to evaluate whether these newly generated strains adapt differently to donkey tetherin (do-tetherin) based on their virulence. We found that do-tetherin exerts an inhibition on the release of the viral particles produced by all three strains, albeit with varying intensity: EIAVDV < EIAVDLV < EIAVFDDV. Additionally, all three EIAV strains could counteract the restriction mediated by do-tetherin via their envelope proteins (Env) with varying strength: EIAVDV > EIAVDLV > EIAVFDDV. These results indicate that donkey tetherin is involved in shaping of EIAV evolution during serial passage.

Identification and characterization of a common B-cell epitope on EIAV capsid proteins.

The equine infectious anemia virus (EIAV) capsid protein (p26) is one of the major immunogenic proteins during EIAV infection and is widely used for the detection of EIAV antibodies in horses. However, few reports have described the use of EIAV-specific monoclonal antibodies (MAbs) in etiological and immunological detection. Previously, we developed an antigen capture enzyme-linked immunosorbent assay (AC-ELISA) for the quantification of the EIAV p26 protein level. However, the epitopes recognized by the MAbs were not identified, and the utilization of the MAbs needs to be evaluated. In this study, we characterized two monoclonal antibodies (9H8 and 1G11 MAbs) against EIAV p26. Two B-cell epitopes are located in amino acid residues, 73NLDKIAEE81 (HE) and 199KNAMRHLRPEDTLEEKMYAC218 (GE) for the 9H8 and 1G11 MAbs, respectively. The 1G11 epitope (GE) varied among viruses isolated worldwide but can be recognized by anti-EIAV sera from different regions, including China, the USA, and Argentina. Meanwhile, 1G11 MAb could react with the mutants of almost all the EIAV strains. Furthermore, we found that the histidine at position 204 (H204), leucine at position 205 (L205), and aspartic acid at position 209 (D209) of EIAV p26 individually played pivotal roles in binding with the 1G11 MAb. Our results revealed that the GE peptide might be a common B-cell binding epitope of EIAV antibodies. This is also the first report to identify a broad-spectrum monoclonal antibody (1G11) against p26 of EIAV. These findings may provide a useful basis for the development of new diagnostic assays for EIAV.

Development and evaluation of a new lateral flow assay for simultaneous detection of antibodies against African Horse Sickness and Equine Infectious Anemia viruses.

African horse sickness (AHS) and equine infectious anemia (EIA) are both notifiable equid specific diseases that may present similar clinical signs. Considering the increased global movement of horses and equine products over the past decades, together with the socio-economic impact of previous AHS and EIA outbreaks, there is a clear demand for an early discrimination and a strict control of their transmission between enzootic and AHS/EIA-free regions. Currently, the individual control and prevention of AHS or EIA relies on a series of measures, including the restriction of animal movements, vector control, and the use of several laboratory techniques for viral identification, amongst others. Despite being widely employed in surveillance programmes and in the control of animal movements, the available serological assays can only detect AHS- or EIA-specific antibodies individually. In this work, a duplex lateral flow assay (LFA) for simultaneous detection and differentiation of specific antibodies against AHS virus (AHSV) and EIA virus (EIAV) was developed and evaluated with experimental and field serum samples. The duplex LFA was based on the AHSV-VP7 outer core protein and the EIAV-P26 major core protein. The results indicated that the duplex LFA presented a good analytical performance, detecting simultaneously and specifically antibodies against AHSV and EIAV. The initial diagnostic evaluation revealed a good agreement with results from the AHS and EIA tests prescribed by the OIE, and it highlighted the usefulness of the new AHSV/EIAV duplex LFA for an on-field and point-of-care first diagnosis.

Amino acid mutations in the env gp90 protein that modify N-linked glycosylation of the Chinese EIAV vaccine strain enhance resistance to neutralizing antibodies.

The Chinese EIAV vaccine is an attenuated live virus vaccine obtained by serial passage of a virulent horse isolate (EIAVL) in donkeys (EIAVD) and, subsequently, in donkey cells in vitro. In this study, we compare the env gene of the original horse virulent virus (EIAVL) with attenuated strains serially passaged in donkey MDM (EIAVDLV) and donkey dermal cells (EIAVFDDV). Genetic comparisons among parental and attenuated strains found that vaccine strains contained amino acid substitutions/deletions in gp90 that resulted in a loss of three potential N-linked glycosylation sites, designated g5, g9, and g10. To investigate the biological significance of these changes, reverse-mutated viruses were constructed in the backbone of the EIAVFDDV infectious molecular clone (pLGFD3). The resulting virus stocks were characterized for replication efficiency in donkey dermal cells and donkey MDM, and were tested for sensitivity to neutralization using sera from two ponies experimentally infected with EIAVFDDV. Results clearly show that these mutations generated by site-directed mutagenesis resulted in cloned viruses with enhanced resistance to serum neutralizing antibodies that were also able to recognize parental viruses. This study indicates that these mutations played an important role in the attenuation of the EIAV vaccine strains.

Health and epidemiological approaches of Trypanosoma evansi and equine infectious anemia virus in naturally infected horses at southern Pantanal.

Equine infectious anemia virus (EIAV) and Trypanossoma evansi are endemic in Brazilian Pantanal Biome, an important area for livestock production. In this sense, we evaluated the epidemiological single and co-infection effects of T. evansi and EIAV in naturally infected horses in the southern Pantanal wetland by serological tests and hematological assays. Both higher seroprevalence and heath poor condition of the sampled animals were associated with differences in horse management between farms. We found that the negative animals for both infectious agents (NN) represented the major group in F1 (37%), and the smallest group in F2 (19%). Furthermore, we recorded higher EIAV seroprevalence (56%) in F2, compared to F1 (38%). We observed that T. evansi infection was mostly related to young horses, as seen by their higher seroprevalence, ranging from 70.7% in the beginning of the rainy season to 81% in the end of flood period, in comparison with the values of 42% and 68%, respectively, in working animals. on the other hand, working animals showed a higher seroprevalence for EIAV (48%) in both seasons than young horses. We observed that the management of working horses could be a risk factor of EIAV infection. On the other hand, as T. evansi is maintained in the study region by many species of wild mammals, the mechanical transmission through blood-sucking vectors ensures the infection to horses since early. Our results showed that single or co-infection by EIAV and T. evansi caused different degree of anemia in the infected animals. Moreover, the health of horses in Brazilian Pantanal is also influenced by differences in horse management and environmental circumstances.

Evolution of equine infectious anaemia in naturally infected mules with different serological reactivity patterns prior and after immune suppression.

Information on equine infectious anaemia (EIA) in mules, including those with an equivocal reaction in agar gel immunodiffusion test (AGIDT), is scarce. For this, a study was conducted to evaluate the clinical, viral loads and pathological findings of two groups of naturally infected asymptomatic mules, respectively with a negative/equivocal and positive AGIDT reactivity, which were subjected to pharmacological immune suppression (IS). A non-infected control was included in the study that remained negative during the observation period. Throughout the whole study, even repeated episodes of recrudescence of EIA were observed in 9 infected mules, independently from their AGIDT reactivity. These events were generally characterised by mild, transient alterations, typical of the EIA acute form represented by hyperthermia and thrombocytopenia, in concomitance with viral RNA (vRNA) peaks that were higher in the Post-IS period, reaching values similar to those of horses during the clinical acute phase of EIA. Total tissue viral nucleic acid loads were greatest in animals with the major vRNA activity and in particular in those with negative/equivocal AGIDT reactivity. vRNA replication levels were around 10-1000 times lower than those reported in horses, with the animals still presenting typical alterations of EIA reactivation. Macroscopic lesions were absent in all the infected animals while histological alterations were characterised by lymphomonocyte infiltrates and moderate hemosiderosis in the cytoplasm of macrophages. On the basis of the above results, even mules with an equivocal/negative AGIDT reaction may act as EIAV reservoirs. Moreover, such animals could escape detection due to the low AGIDT sensitivity and therefore contribute to the maintenance and spread of the infection.

Serologically silent, occult equine infectious anemia virus (EIAV) infections in horses.

Molecular and serological techniques for Equine Infectious Anemia Virus (EIAV) diagnosis were compared using samples from 59 clinically normal horses stabled on five farms in the Santa Fe Province of Argentina. Of these 26 (44.1%) were positive in official AGID tests and/or gp45/gp90-based ELISA. Surprisingly 18 of the 33 seronegative horses were positive in a PCR against viral sequences encoding gp45 (PCR-positive/AGID-negative) with all but one remaining EIAV-antibody negative throughout a two year observation period. The gp45 PCR results are supported by fact that 7/18 of these horses were positive in the Office International des Epizooties (OIE) recommended EIAV gag gene specific PCR plus 2 of this 7 also reacted in a PCR directed predominantly against the 5' untranslated region of the viral genome. Furthermore sufficient quantities of serum were available from 8 of these horses to verify their seronegative status in sensitive Western Blot tests and demonstrate by ELISA the absence of EIAV-specific antibodies was not attributable to abnormalities in total IgG concentration. Studies involving 7 of the PCR-positive/AGID-negative horses to measure lymphocyte proliferation in the presence of PHA showed no significant differences between this group and control animals. In addition, lymphocytes from 2 of these 7 horses responded to peptides derived from gp90 and gp45. Together these results demonstrate that apparently clinically normal horses with no gross signs of immunodeficiency in terms of total IgG concentration or T helper-cell function can remain seronegative for at least 24 months while harboring EIAV specific nucleic acid sequences.

Genetic Evolution during the development of an attenuated EIAV vaccine.

BACKGROUND: The equine infectious anemia virus (EIAV) vaccine is the only attenuated lentiviral vaccine applied on a large scale that has been shown to be effective in controlling the prevalence of EIA in China. This vaccine was developed by successive passaging of a field-isolated virulent strain in different hosts and cultivated cells. To explore the molecular basis for the phenotype alteration of this vaccine strain, we systematically analyzed its genomic evolution during vaccine development. RESULTS: Sequence analysis revealed that the genetic distance between the wild-type strain and six representative strains isolated from key development stages gradually increased with the number of passages. Env gene, but not gag and pol, showed a clear evolutionary flow similar to that of the whole genomes of different generations during the attenuation. Stable mutations were identified in multiple regions of multiple genes along with virus passaging. The adaption of the virus to the growth environment of cultured cells with accumulated genomic and genetic variations was positively correlated with the reduction in pathogenicity and rise of immunogenicity. Statistical analyses revealed significant differences in the frequency of the most stable mutations between in vivo and ex vivo-adapted strains and between virulent and attenuated strains. CONCLUSIONS: These data indicate that EIAV evolution during vaccine development generated an accumulation of mutations under the selective drive force, which helps to better understand the molecular basis of lentivirus pathogenicity and immunogenicity.

Infection with equine infectious anemia virus vaccine strain EIAVDLV121 causes no visible histopathological lesions in target organs in association with restricted viral replication and unique cytokine response.

The live equine infectious anemia virus (EIAV) vaccine strain EIAVDLV121 was developed by in vitro attenuation of a virulent strain, EIAVLN40, in the 1970s, and it has been demonstrated to induce protective immunity under laboratory and natural EIAV infection conditions. The detailed biological features of this attenuated virus remain to be further investigated. Experimental inoculation with EIAVDLV121 did not result in clinical symptoms even with immunosuppressive treatment in our previous studies. Here, we further investigated whether the replication of the vaccine strain EIAVDLV121 in experimentally infected horses causes histopathological lesions to develop in the targeted organs. Both the lungs and the spleen have been demonstrated to support EIAV replication. By evaluating the gross macroscopic and histological changes, we found that EIAVDLV121 did not cause detectable histopathological lesions and that it replicated several hundred times more slowly than its parental virulent strain, EIAVLN40, in tissues. Immunochemical assays of these tissues indicated that the primary target cells of EIAVDLV121 were monocytes/macrophages, but that EIAVLN40 also infected alveolar epithelial cells and vascular endothelial cells. In addition, both of these viral strains promoted the up- and down-regulation of the expression of various cytokines and chemokines, implicating the potential involvement of these cellular factors in the pathological outcomes of EIAV infection and host immune responses. Taken together, these results demonstrate that the EIAV vaccine strain does not cause obvious histopathological lesions or clinical symptoms and that it induces a unique cytokine response profile. These features are considered essential for EIAVDLV121 to function as an effective live vaccine.

Structural Illumination of Equine MHC Class I Molecules Highlights Unconventional Epitope Presentation Manner That Is Evolved in Equine Leukocyte Antigen Alleles.

MHC class I (MHC I)-restricted virus-specific CTLs are implicated as critical components in the control of this naturally occurring lentivirus and in the protective immune response to the successfully applied attenuated equine infectious anemia virus vaccine in the horse. Nevertheless, the structural basis for how the equine MHC I presents epitope peptides remains unknown. In this study, we investigated the binding of several equine infectious anemia virus-derived epitope peptides by the ability to refold recombinant molecules and by thermal stability, and then by determining the x-ray structure of five peptide-MHC I complexes: equine MHC class I allele (Eqca)-N*00602/Env-RW12, Eqca-N*00602/Gag-GW12, Eqca-N*00602/Rev-QW11, Eqca-N*00602/Gag-CF9, and Eqca-N*00601/Gag-GW12. Although Eqca-N*00601 and Eqca-N*00602 differ by a single amino acid, Eqca-N*00601 exhibited a drastically different peptide presentation when binding a similar CTL epitope, Gag-GW12; the result makes the previously reported function clear to be non-cross-recognition between these two alleles. The structures plus Eqca-N*00602 complexed with a 9-mer peptide are particularly noteworthy in that we illuminated differences in apparent flexibility in the center of the epitope peptides for the complexes with Gag-GW12 as compared with Env-RW12, and a strict selection of epitope peptides with normal length. The featured preferences and unconventional presentations of long peptides by equine MHC I molecules provide structural bases to explain the exceptional anti-lentivirus immunity in the horse. We think that the beneficial reference points could serve as an initial platform for other human or animal lentiviruses.

Insights into vaccine development for acquired immune deficiency syndrome from crystal structures of human immunodeficiency virus-1 gp41 and equine infectious anemia virus gp45.

An effective vaccine against acquired immune deficiency syndrome is still unavailable after dozens of years of striving. The glycoprotein gp41 of human immunodeficiency virus is a good candidate as potential immunogen because of its conservation and relatively low glycosylation. As a reference of human immunodeficiency virus gp41, gp45 from equine infectious anemia virus (EIAV) could be used for comparison because both wild-type and vaccine strain of EIAV have been extensively studied. From structural studies of these proteins, the conformational changes during viral invasion could be unveiled, and a more effective acquired immune deficiency syndrome vaccine immunogen might be designed based on this information.