| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
,
1 State Key Laboratory for Infectious Disease Prevention and Control, National Center for AIDS/STD Control and Prevention, China CDC, Beijing 100050, China
2 Harbin Veterinary Research Institute, Harbin 150086, China
Correspondence
Rongxian Shen
Yiming Shao
yshao{at}bbn.cn
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
39 °C), which coincided with higher viral load (106–107 copies ml–1) and lower cytokine production (<104 copies per 107 PBMCs). The results indicate that high levels of EIAV-specific cytokines induced by the attenuated EIAV vaccine may contribute to the protective immune response against EIA disease. Supplementary tables showing the primers used for this study are available in JGV Online.
These authors contributed equally to this paper. 
Present address: Shanghai Municipal Center for Disease Control & Prevention, Shanghai 200336, China.
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
). DLV is the only retrovirus attenuated vaccine used on a large scale in the world to date. It provides a unique natural model system. Elucidation of the DLV attenuated mechanism and immunological protective mechanisms may aid in the development of vaccines for other retroviruses, including HIV. In previous studies, we have identified several key mutation sites in the DLV sequence which are likely to contribute to the attenuation (Liang et al., 2006; Shen et al., 2006). However, the immunological protective mechanism of EIAV attenuated vaccine DLV was not clear.
It was shown that neutralizing antibody responses developed about 30–40 days post-infection and matured over a period of several months (Kono et al., 1973; Rwambo et al., 1990; Hammond et al., 1997). The period of immune system maturation after vaccination with live-attenuated vaccine is at least 6 months in horses (Shen et al., 1979; Li et al., 2003). The neutralizing antibodies are primarily directed toward the EIAV envelope. Given the relatively slow development of neutralizing antibodies, cellular immune responses are likely to be important in combating viruses in the early stage of EIAV infection. Studies showed that cellular immunity could significantly contribute to the control of lentivirus infection (Jin et al., 1999; Ogg et al., 1999; Schmitz et al., 1999). The cytotoxic T-lymphocytes (CTL) response is considered to play a critical role in the control of HIV replication (Goulder & Watkins, 2004). EIAV-specific CD8+ and CD4+ CTL have been identified in horses recently infected with EIAV and memory CTL are present in EIAV-infected healthy carrier horses (McGuire et al., 2004), indicating that the CTL response may associate with the control of virus replication. However, it remains unknown whether this is the case in our attenuated EIAV vaccine model.
Proinflammatory cytokines are the central mediators of both innate and adaptive immunity and modulation of these cytokines has been shown to alter anti-HIV-1 reactivity in vitro (Connolly et al., 2005). Because the Th-1 cytokines gamma interferon (IFN-
), interleukin (IL)-2 and -12 have been widely used to gauge the activities of cellular immunity, methods were established to measure the production of these cytokines in horses (Wang et al., 2005) to determine cellular immunity.
In this study, horses were immunized and then challenged with EIAV wild-type strain LN. The resulting cytokine production and viral load levels in peripheral blood mononuclear cells (PBMCs) of vaccinated and non-vaccinated control groups were compared.
|
METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
). DL cell cultures were prepared from heparinized peripheral blood as equine MDM (monocyte/macrophage differentiation-maturation system) cells. The minimal infectious dose of DLV was determined to be 1x104 TCID50 by limiting dilution assay with DL cells.
Horses, clinical evaluation and sample collection.
All horses used in these studies were thoroughbreds. Prior to the experiment, serum samples from all horses were tested twice by the agar gel immunodiffusion test (VMRD) to ensure that they were seronegative for EIAV infection (Pare & Simard, 2004). They were housed in screened box stalls to exclude haematophagous insects and all animal handling protocols were approved by the Animal Management Committee of Chinese Harbin Veterinary Research Institute. Rectal temperature and clinical status were recorded twice daily. Samples of plasma and whole blood were collected every 2 weeks in the first 3 months and then every month thereafter. Samples were also collected during each febrile episode (rectal temperature 39 °C). The plasma samples were stored at –80 °C for viral load detection. Clinical episodes of equine infectious anaemia (EIA) were defined when thrombocytopenia (<100 000 platelets µl–1) occurred with fever, concurrent with detectable plasma viral RNA.
Vaccination procedures.
The animals were randomized into four groups: naive healthy horses (A2 and A5) inoculated with 3 ml supernatant of normal DL culture as negative controls (Group I), DLV vaccination followed by a virulent EIAV challenge (A3, A6, A7 and A8) (Group II), virulent EIAV challenge without DLV vaccination (A1, A4, A10 and A12) (Group III) and naturally EIAV-infected horses (C1, C2, C65 and C66) as positive controls (Group IV). The Group IV horses were discovered when the horses were screened for EIAV infection by agar gel immunodiffusion test prior to the study. We arbitrarily selected four seropositive horses for EIAV infection that had had no clinical presentations and fever episodes in the last 3 months. The important regions of the viral genome, including gag, pol and env, were sequenced. Sequencing data showed that the EIAV strain naturally present in horses had nearly 70 % similarity with LN strain in amino acid sequence. The dose of DLV vaccination was 4x105 TCID50 and that of virulent EIAV LN challenge was 105 TCID50. All animals were subcutaneously injected with vaccine or challenge strain.
Viral RNA purification.
All samples from the same time point were processed in parallel to minimize the difference potentially caused by the operator. Viral RNA was isolated from 140 µl EDTA-anticoagulated plasma by using a QIAamp Viral RNA kit (Qiagen) and treated with DNase I on a spin column (DNase I set; Qiagen). All procedures strictly adhered to the manufacturer's instructions. RNA was finally eluted in 60 µl AVE buffer and analysed immediately.
Quantification of plasma virus RNA.
Real-time RT-PCR was used to determine plasma viral load by amplification of the EIAV gag gene (Liang et al., 2006). Briefly, the following primers were used for real-time RT-PCR using a QuantiTect Probe RT-PCR kit (Qiagen): forward primer, 5'-CAGATTGCTGTCTCAGATAAA-3'; reverse primer, 5'-GTGTCTGTCAGGAATTTAGTT-3'; and TaqMan probe (Applied Biosystems), 5'-FAM-TCAGCCGGATGTCCCTCACT-TAMRA-3'. An RNA standard template was made by cloning the partial gag DNA segment from pFD3 (nt 1144–1533) into pGEM-T Easy vector (Promega), followed by in vitro transcription. Data analysis was conducted with Sequence Detection software version 1.9 (Perkin-Elmer).
Equine cytokine production detection.
Specific primers of horse IFN- (Curran et al., 1994; GenBank accession no. D28520
[GenBank]
), IL-2 (accession no. X69393
[GenBank]
) and IL-12 IP40 subunit (accession no. Y11130
[GenBank]
) are listed in Supplementary Tables S1 and S2 (available in JGV Online).
Preparation of cytokine RNA.
Total RNA was isolated from healthy and vaccinated horse PBMCs with an RNeasy mini kit (Qiagen), according to the manufacturer's instructions. Briefly, freshly isolated horse PBMCs (1x107) were disrupted by adding 600 µl Buffer RLT (Qiagen). The lysate was homogenized for 30 s using a rotor–stator homogenizer, and 1 vol. 70 % ethanol was added. Then, 700 µl sample was transferred to an RNeasy spin column and centrifuged for 15 s at 10 000 r.p.m. (8500 g); the RNA was eluted with 50 µl RNase-free water.
After total RNA was prepared, the quality of the RNA samples was examined by agarose-formaldehyde electrophoresis and ethidium bromide staining. The respective rRNAs should appear as clear bands or peaks. The apparent ratio of 28S rRNA to 18S rRNA should be approximately 2 : 1. The RNA samples were also examined by absorbance measurement to assess the RNA recovery efficiency.
RNA standard preparation.
First-strand cDNA was primed in a 10 µl DEPC H2O solution containing 1 µg total RNA isolated from healthy horse PBMCs, 50 pmol oligo(dT) primer and 1.5 U reverse transcriptase. Reaction conditions were as follows: 65 °C for 5 min, then 25 °C for 10 min. Full-length DNA fragments of IFN- (521 bp), IL-2 (632 bp) and IL-12 P40 subunit (669 bp) were separately amplified by PCR in 20 µl reactions containing 4 µl PCR reaction buffer (5x), 0.4 µM forward primer, 0.4 µM reverse primer, 0.5 mM dNTP, 4 units Taq DNA polymerase (Qiagen) and 1 µl cDNA. Reaction conditions were as follows: 95 °C for 2 min, followed by 30 cycles at 94 °C for 20 s, 56 °C for 20 s and 72 °C for 30 s, and finally 72 °C for 5 min. The PCR products were purified and cloned into pBluescript II SK vector (Stratagene) individually for RNA transcription in vitro. The RNA transcripts were used as RNA standards.
Detection of the corresponding copy numbers of cytokines.
IFN-, IL-2 and IL-12 mRNAs from horses inoculated with EIAV virulent or attenuated strains were detected using real-time RT-PCR. The 50 µl reaction solutions contained 25 µl RT-PCR Master Mix (2x; Applied Biosystems), 0.4 µM forward primer, 0.4 µM reverse primer, 0.2 µM Taqman probe, 0.5 µl RT mix and 10 µl RNA sample or RNA standard. Reactions were performed in triplicate. Cycling conditions were 48 °C for 30 min and 95 °C for 10 min, followed by 40 cycles at 95 °C for 15 s and 60 °C for 1 min.
The cycle threshold (Ct) value for each sample was calculated by determining the point at which the fluorescence exceeded a threshold limit (10 times the standard deviation of the baseline as determined between cycles 3 and 15). Standard curves proportional to the log of the starting amount of nucleic acid were produced according to that Ct value. Detection and data analysis were performed with the ABI Prism 5700 SDS program.
Statistical analysis.
Two-tailed t-test and ranks test were used for normal and abnormal distributions, respectively. An alpha level of 0.05 was used.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
Group I and II horses remained asymptomatic for EIA. This was demonstrated by lack of fever episodes and thrombocytopenia, which are considered to be sensitive indicators of EIA. The absence of detectable clinical symptoms after DLV vaccination was associated with the relatively low level of plasma viral loads, ranging from undetectable to 103 copies (ml of plasma)–1 (data not shown). The sustained low-level virus replication may be helpful in driving the maturation of immune responses to establish enduring, broad protective immunity.
Eight months later, Group II horses were challenged with virulent EIAV LN, all horses in this group survived the observation period (Fig. 1). The horses showed no temperature increase except for one febrile episode (39–42 °C) in one animal (A3), which was concurrent with a lower platelet count (91 750 platelets µl–1) that returned to normal 3 days later. The others remained asymptomatic for EIA. In the first 3 months post-LN challenge, the viral loads of horses A3, A6, A7 and A8 were consistently less than 1x103 copies ml–1, except A3 which reached 1x105 copies ml–1 during the fever episode. Thereafter, the viral load was too low to be detectable (Fig. 2a).
|
|
). Horse A10 died on day 219. The reason for the death of A10 remains unknown since no pathological examination was accessible at that time and may not be related to EIA since horse A10 was not febrile and did not have an obvious thrombocytopenia at the time of death.
The lowest platelet counts were about 45 000 platelets µl–1 for A1 and A12 and 65 000 platelets µl–1 for A4 and A10 during the fever episodes. The plasma viral RNA levels reached 108 copies (ml of plasma)–1 during fever episodes, and were in excess of 103 copies ml–1 even in the interval between fever episodes (Fig. 2b).
Cytokine production
As shown in Fig. 3(a), the mean baselines of IFN-, IL-2 and IL-12 in PBMCs were 3.7x104, 4.99x103 and 4.03x105 copies per 107 PBMCs, respectively, for horses without any treatment; and 4.05x105, 5.39x104 and 1.3x106 copies per 107 PBMCs, respectively, for Group IV. The absolute value of IL-12 was highest among the three cytokines in both groups. However, the magnitude increase of IL-12 (3-fold) elicited by EIAV natural infection was much lower than that of IFN- and IL-2, which were almost 11-fold. A similar trend was also observed in the DLV-vaccinated group. The production of all three cytokines increased steadily for 2 weeks and reached a plateau 2–3 months post-DLV vaccination. The corresponding values for IFN-, IL-2 and IL-12 were 7.33x106, 5.84x105 and 2.16x106 copies per 107 PBMCs. These represent significant increases above the baseline (198-fold, 117-fold and 5-fold, respectively; P<0.01) and above that of Group IV (18-fold, 10-fold and 2-fold, respectively; P<0.01).
|
) and peaked (107–108 copies per 107 PBMCs) 1 month post-LN infection; thereafter, the cytokine production gradually declined. Interestingly, while both IFN- and IL-12 levels dropped to the baseline level 3 months post-LN challenge, IL-2 production retained an approximately 10-fold baseline level, suggesting that IL-2 level may be an important indicator for protection.
All horses without DLV vaccination developed acute or chronic disease after challenge with virulent EIAV. There were two different patterns of disease progression (Fig. 4). One pattern was rapid progression to EIA and death within 3 weeks, such as in horses A1 and A12; the second was slow progression as exemplified by horse A4. During the primary acute infection period, a transient increase of cytokine production of IFN- and IL-12 was observed in horses A1, A10 and A12 but not in horse A4. It was followed by a decrease when the fever episode occurred (2 weeks for A1 and A12 to develop the fever episode and 65 days for A10). IL-2 production increased post-infection only in horse A10, which coincided with the absence of clinical episode within the first 65 days post-infection and prolonged survival. A gradual recovery of cytokine production in horses A4 and A10 occurred coincidentally with the decrease of viral loads and resolution of fever.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
). The mechanism by which the attenuated EIAV vaccine protects against viral challenge by the diverse virulent EIAV strains remains to be determined. In this study, the previous observation that DLV vaccination could protect against EIA disease was partially confirmed. Three out of four vaccinated horses were fully protected from EIA disease and did not show symptoms. The 100 % protected horses (A6, A7 and A8) were found to maintain normal temperatures and low levels of plasma viral loads. Only one horse (A3) showed a spike of high fever and higher viral load (105 copies ml–1). Non-vaccinated horses challenged with LN, however, developed typical EIA. A1 and A12 died after only a single clinical episode, A4 and A10 died after several fever episodes though A10 was afebrile at the time of death. To elucidate the protective mechanism, horse temperature, plasma viral loads and EIAV-specific cytokine production were monitored. Viral load was highest in the group of horses challenged with LN without DLV vaccination [about 107–108 copies (ml plasma)–1 during a febrile episode and greater than 103 copies ml–1 between febrile episodes].
Significantly increased expression of IFN-, IL-2 and IL-12 was observed after both DLV vaccination and the following virulent EIAV LN challenge. Overall, the cytokine data indicated that: (i) DLV vaccination resulted in a significant increase in cytokine production compared to baseline and to negative controls, which was also higher than that seen in naturally EIAV-infected horses; (ii) IFN- and IL-12 returned to a level similar to baseline 3 months post-LN challenge; (iii) IL-2 levels remained relatively high and constant after LN challenge in the DLV-vaccinated horses and may be an important predictor for protection; (iv) cytokine levels were either not sustained or fluctuated at a lower level in non-vaccinated horses after LN challenge.
It has been demonstrated that antibody responses mature by increasing in avidity and conformational dependence following infection with EIAV (Hammond et al., 1997) and may play a role in protection from disease or infection. In the present study, neutralizing antibodies were also detected in the sera of horses vaccinated with DLV. These results suggest that neutralizing antibodies elicited by DLV may play an important role in the control of EIAV infection of LN strain (Shen et al., 1979; and unpublished data). The mechanism of the humoral immune response elicited by DLV is currently under investigation.
The elucidation of these immunological protective mechanisms should provide a foundation for designing new EIAV vaccines, which may also lead to more effective vaccines for other lentiviral infections such as HIV.
|
ACKNOWLEDGEMENTS |
|---|
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
Curran, J. A., Argyle, D. J., Cox, P., Onions, D. E. & Nicolson, L. (1994). Nucleotide sequence of the equine interferon gamma cDNA. DNA Seq 4, 405–407.[Medline]
Goulder, P. J. & Watkins, D. I. (2004). HIV and SIV CTL escape: implications for vaccine design. Nat Rev Immunol 4, 630–640.[CrossRef][Medline]
Hammond, S. A., Cook, S. J., Lichtenstein, D. L., Issel, C. J. & Montelaro, R. C. (1997). Maturation of the cellular and humoral immune responses to persistent infection in horses by equine infectious anemia virus is a complex and lengthy process. J Virol 71, 3840–3852.[Abstract]
Jin, X., Bauer, D. E., Tuttleton, S. E., Lewin, S., Gettie, A., Blanchard, J., Irwin, C. E., Safrit, J. T., Mittler, J. & other authors (1999). Dramatic rise in plasma viremia after CD8(+) T cell depletion in simian immunodeficiency virus-infected macaques. J Exp Med 189, 991–998.
Kono, Y., Kobayashi, K. & Fukunaga, Y. (1973). Antigenic drift of equine infectious anemia virus in chronically infected horses. Arch Gesamte Virusforsch 41, 1–10.[CrossRef][Medline]
Li, F., Craigo, J. K., Howe, L., Steckbeck, J. D., Cook, S., Issel, C. & Montelaro, R. C. (2003). A live attenuated equine infectious anemia virus proviral vaccine with a modified S2 gene provides protection from detectable infection by intravenous virulent virus challenge of experimentally inoculated horses. J Virol 77, 7244–7253.
Liang, H., He, X., Shen, R. X., Shen, T., Tong, X., Ma, Y., Xiang, W. H., Zhang, X. Y. & Shao, Y. M. (2006). Combined amino acid mutations occurring in the envelope closely correlate with pathogenicity of EIAV. Arch Virol 151, 1387–1403.[CrossRef][Medline]
McGuire, T. C., Fraser, D. G. & Mealey, R. H. (2004). Cytotoxic T lymphocytes in protection against equine infectious anemia virus. Anim Health Res Rev 5, 271–276.[CrossRef][Medline]
Ogg, G. S., Jin, X., Bonhoeffer, S., Dunbar, P. R., Nowak, M. A., Monard, S., Segal, J. P., Cao, Y., Rowland-Jones, S. L. & other authors (1998). Quantitation of HIV-1-specific cytotoxic T lymphocytes and plasma load of viral RNA. Science 279, 2103–2106.
Pare, J. & Simard, C. (2004). Comparison of commercial enzyme-linked immunosorbent assays and agar gel immunodiffusion tests for the serodiagnosis of equine infectious anemia. Can J Vet Res 68, 254–258.[Medline]
Rwambo, P. M., Issel, C. J., Adams, W. V., Jr, Hussain, K. A., Miller, M. & Montelaro, R. C. (1990). Equine infectious anemia virus (EIAV) humoral responses of recipient ponies and antigenic variation during persistent infection. Arch Virol 111, 199–212.[CrossRef][Medline]
Schmitz, J. E., Kuroda, M. J., Santra, S., Sasseville, V. G., Simon, M. A., Lifton, M. A., Racz, P., Tenner-Racz, K., Dalesandro, M. & other authors (1999). Control of viremia in simian immunodeficiency virus infection by CD8+ lymphocytes. Science 283, 857–860.
Shen, R. X., Xu, Z. D., He, X. S. & Zhang, S. X. (1979). Study on immunological methods of equine infectious anemia. China Agric Sci 4, 1–15 (in Chinese).
Shen, T., Liang, H., Tong, X., Fan, X., He, X., Ma, Y., Xiang, W., Shen, R., Zhang, X. & Shao, Y. (2006). Amino acid mutations of the infectious clone from Chinese EIAV attenuated vaccine resulted in reversion of virulence. Vaccine 24, 738–749.[CrossRef][Medline]
Wang, Y., Wei, L. L., Zhang, X. Y., Wu, D. L., Xiang, W. H., Wang, X. J., Lv, X. L., Shen, R. X. & Shao, Y. M. (2005). Evaluation of IL-2 expressing level in PBMC of EIAV vaccinated horses. China Biotechnology 25, 55–59 (in Chinese).
Received 26 July 2006;
accepted 5 November 2006.
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
| J MED MICROBIOL | ALL SGM JOURNALS | |
, 
, and – indicate horse viral load, platelet and temperature, respectively
) and EIAV naturally infected horses (
) are shown at left (a) and DLV vaccinated horses post-viral challenge are shown at right (b). The x-axis indicates days post-immunization (a) and challenge (b). The y-axis indicates cytokine production (copies per 107 PBMCs).