Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA1
Department of Veterinary Science, Gluck Equine Research Center, University of Kentucky, Lexington, KY 40546, USA2
Author for correspondence: Ronald Montelaro. Fax +1 412 383 8859. e-mail rmont{at}pitt.edu
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Abstract |
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Sequence analysis of EIAV envelope variation during progressive disease cycles demonstrates distinct envelope variants with each wave of viraemia (Leroux et al., 1997 , 2001
; Lichtenstein et al., 1996
; Payne et al., 1987a
). The emergence of novel EIAV populations with sequential disease episodes indicates the cyclical nature of chronic disease that results from sequential production and selection of viral antigenic variants temporarily escaping established host immune responses. The principal site of antigenic variation is the envelope surface unit glycoprotein gp90 (Payne et al., 1987b
; Rwambo et al., 1990a
; Zheng et al., 1997
). The genomic variation of gp90 has been analysed in order to define conserved and variable protein domains (Leroux et al., 1997
, 2001
; Payne et al., 1989
), as observed with other animal and human lentiviruses (Greene et al., 1993
; Simmonds et al., 1990
; Starcich et al., 1986
; Suarez & Whetstone, 1995
). While the role of envelope variation in altering in vitro antigenic properties (e.g. neutralization sensitivity) has been well documented for over 20 years, there is, to date, no analysis of the effects of EIAV gp90 variation on immunogenicity in experimentally infected equids.
Studies indicate that control of EIAV replication and disease is specifically mediated by host immune responses controlling virus infection to subclinical levels and not by attenuation of the virus during persistent infection (Issel et al., 1982 ; Kono et al., 1976a
; Tumas et al., 1994
). Immune management of EIAV replication and disease is, evidently, the result of the evolution of both humoral and cellular immune responses (Hammond et al., 1997
). The predominant humoral response, including virus-neutralizing antibodies to EIAV infection, is directed against gp90 (Rwambo et al., 1990b
). We defined previously three neutralizing epitopes in gp90 (Ball et al., 1992
; Hussain et al., 1987
, 1988
). Epitopes DNT and ENT are localized to the V3 domain of gp90, while epitope CNT is associated with the V5 domain. The V3 region has been designated the principal neutralizing domain (PND) (Ball et al., 1992
).
As part of a previous comprehensive investigation of the immune responses to virus infection and viral genomic variation in four ponies experimentally infected with EIAVPV (Hammond et al., 1997 , 2000
; Leroux et al., 1997
, 2001
), we isolated and characterized a novel serum neutralization-resistant variant containing a 14 amino acid deletion in the PND segment of gp90 (EIAVPV564
PND). The entire envelope of this variant was cloned into a reference strain of EIAV to generate a chimeric clone to further analyse the effects of the PND deletion on in vitro virus replication (Leroux et al., 1997
). These studies indicated that the PND deletion did not affect in vitro replication properties as compared to the reference EIAVPV strain. However, EIAV
PND was not neutralized in cell culture by homologous or autologous immune serum from the four experimentally infected ponies or other reference-neutralizing immune serum, all displaying high neutralizing activity of the reference strain EIAVPV (Leroux et al., 1997
). In light of the marked neutralization-resistant phenotype of the EIAV
PND variant, we sought to examine in an experimental infection the immunogenicity of this variant envelope and the role of host immune responses in controlling the replication of EIAV
PND.
To elucidate the effects of the deleted PND on virus replication and immunogenicity during experimental infections, two mixed-breed ponies (animals #599 and #672) were inoculated intravenously with 1x103 TCID50 EIAVPND. The infected ponies remained asymptomatic for EIA throughout a 14-month observation period (Fig. 1
). Virus replication during the initial acute infection, around 30 days post-infection (p.i.), reached approximately 1x106 plasma RNA copies/ml, then rapidly declined to steady-state levels of about 1x104 copies/ml for the following 13 months. We previously demonstrated that clinical EIA is typically associated with replication levels of at least 1x107 plasma RNA copies/ml (Hammond et al., 2000
; Leroux et al., 2001
). The apparent lack of disease during the EIAV
PND infection appeared to be due to the control of virus replication to subclinical levels, indicating the ability of the host immune system to effectively target virus in the absence of the immunodominant gp90 PND.
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By 2 days after the last dose of dexamethasone, both ponies developed a classic EIA episode, experiencing a simultaneous drop in platelets and an increase in temperature (Fig. 1). Recrudescence of disease was accompanied by marked increases in plasma viral RNA (Fig. 1
). Virus replication increased during the immune-suppression period from steady-state levels of 1x104 RNA copies/ml (420 days p.i.) to a peak of 1x108 copies/ml (432 days p.i.) (Fig. 1
). Clinical symptoms resolved by day 434, accompanied by a rapid decline in viral RNA levels, presumably reflecting the return of host immune control. Interestingly, steady-state levels of replication following resolution of the disease cycle were at least 100-fold lower than the steady-state levels observed prior to immune suppression. These observations seem to indicate the enhancement of immune control of the persistent virus infection as a result of transient immune suppression.
Concomitant with the resolution of clinical symptoms and decrease in virus load was a marked increase in envelope-specific antibodies accompanied by the appearance of detectable neutralizing antibodies to the infecting EIAVPND strain (Fig. 2
). The envelope-specific serum antibody titre, which had remained at 1x105 for over 1 year, increased to between 1x106 and 1x106·5 following the dexamethasone-induced disease episode (Fig. 2
). This increase in antibody titre was accompanied by the development of neutralizing antibody titres (1:350 to 1:500) against the infecting EIAV
PND strain (Fig. 2
). Serum-neutralization levels observed in the current experimental infections with EIAV
PND after immune suppression were similar to responses observed in experimental infection with the reference EIAVPV strain (Hammond et al., 2000
). However, neutralizing antibody responses appeared highly type-specific for the
PND envelope. Identical serum samples had no detectable neutralizing activity against the reference EIAVPV strain containing prototypic gp90 with a complete PND (Fig. 2
). Taken together, these observations indicate the ability of the host immune system to generate effective neutralizing antibody responses to a highly neutralization-resistant EIAV envelope lacking an intact immunodominant PND in the gp90 protein, implying that immunorecessive determinants outside the PND can in fact serve as primary determinants for neutralizing antibodies in the absence of the PND.
The unexpected development of neutralizing antibodies to the EIAVPND strain post-immune suppression could be caused by either quantitative increases in existing antibody responses to the EIAV
PND envelope or qualitative changes in antibody specificity triggered by envelope variation post-immune suppression. To examine these two possibilities, we characterized and compared viral envelope genomic quasispecies present immediately prior to immune suppression and the population associated with the febrile episode after immune suppression (Fig. 3
). In general, viral envelopes detected pre- and post-immune suppression revealed approximately 34% variation from the envelope sequence of the EIAV
PND inoculum. Importantly, all envelope clones retained a PND-deleted V3 region, with amino acid variations localized to variable domains of the gp90 defined previously. These data demonstrated a similar evolution rate of EIAV envelopes during persistent infection in the presence or absence of detectable serum-neutralizing antibodies, suggesting there are selective pressures by other immune or non-immune host factors on virus variation. Relative to the issue of the antigen specificity of the neutralizing antibody responses, the envelope sequence data revealed that post-immune suppression virus populations contained distinguishing sequence differences compared to viral envelopes present prior to immune suppression. Thus, it is possible that induction of neutralizing antibodies may be due to both qualitative changes in the viral envelope and quantitative changes in viral antigen load due to immune suppression.
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These observations for the first time indicate adaptation of virus-specific host immunity by transient immune suppression of an inapparent EIAV carrier. The basis for this change in antibody specificity is uncertain but raises a number of interesting questions about factors that define steady-state interactions between the immune system and a persistent EIAV infection in inapparent carriers. Interestingly, this inapparent-carrier state was maintained in the absence of detectable neutralizing antibodies, even with an ongoing evolution of gp90 sequences presumably progressively generating de novo antibody responses to evolving virus variants. Transient immune suppression and associated changes in virus-specific immune responses evidently resulted in a further 100-fold lowering of steady-state virus replication levels (1x104 to 1x102 RNA copies/ml). However, it remains to be determined if the enhanced suppression of virus replication is due to introduction of neutralizing antibodies or to modification in other humoral or cellular immune responses.
Studies presented here demonstrate the ability of host cellular and humoral immune responses (excluding neutralizing antibodies) to control a neutralization-resistant EIAV virus in the absence of the gp90 PND. The absence of classic EIA in the first year of infection suggests multiple complementary mechanisms of host immune control of EIAV replication and disease that do not require the presence of the gp90 PND. Control of virus infection could not be attributed to intrinsic attenuation of EIAVPND, as immune suppression with dexamethasone produced high levels of virus replication and a characteristic EIA disease cycle. With the removal of dexamethasone, the immune system rapidly recovered with effective resolution of disease and control of virus replication.
Structured treatment interruption (STI) has been recently proposed as a means of managing HIV-1 infection of patients on highly active anti-retroviral therapy (HAART) (Boyle, 2000 ; Lori et al., 2000
; Ruiz et al., 2000
). STI involves repetitive on-and-off cycles of HAART, which increases virus replication to stimulate virus immunity and, in particluar, the cellular immune response. Initial trials in HIV-1 patients and simian immunodeficiency virus (SIV)-infected monkeys yielded mixed results but indicate a potential to improve virus-specific immunity and lower steady-state virus replication. Effects of transient immune suppression of EIAV-infected inapparent carrier ponies resemble STI studies of HIV-1/SIV infections in that a temporary increase in virus load enhanced virus-specific immunity and reduced virus load. Relevant to our studies with EIAV is a recent report that STI of HIV-1 patients produced increased levels of neutralizing antibodies to primary HIV-1 isolates, in addition to the more frequently observed increase in virus-specific cytotoxic T-cell responses (Montefiori et al., 2001
). Taken together, these findings indicate that temporary managed increases in virus load via removal of antiviral drugs or regulated short-term immunosuppressive therapy allows host immune systems to establish new, more effective responses to established virus infections.
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References |
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Received 25 October 2001;
accepted 4 February 2002.