The determination of in vivo envelope-specific cell-mediated immune responses in equine infectious anemia virus-infected ponies

Distinct from human lentivirus infection, equine infectious anemia virus (EIAV)-infected horses will eventually enter an inapparent carrier state in which virus replication is apparently controlled by adaptive immune responses. Although recrudescence of disease can occur after immune suppression, the actual immune correlate associated with protection has yet to be determined. Therefore, EIAV provides a model for investigating immune-mediated protective mechanisms against lentivirus infection. Here, we have developed a method to monitor EIAV-envelope specific cellular immunity in vivo. An EIA carrier horse with no clinical signs infected 7 years ago and 4 related experimental ponies infected 6 months previously were used in this study. Forty-four 20-mer peptides, representing the entire surface unit protein (gp90) of EIAV, were combined into 14 peptide pools and intradermally injected into the neck of EIAV-infected horses. An identical volume of saline alone was injected into a fifteenth site as a negative control. After 48. h, those sites with palpable infiltrations were measured prior to the collection of 2. mm and 4. mm punch biopsies. Total RNA was extracted from each 2. mm biopsy for determination of CD3 and interferon-γ (IFN-γ) mRNA expression by real-time PCR. The 4. mm skin biopsies were formalin-fixed and paraffin-embedded for immunohistochemistry (IHC) staining for CD3, CD20, CD25 and MAC387 (macrophage marker). Peripheral blood mononuclear cells (PBMC) were obtained prior to the injection and tested for in vitro reactivity against the same peptides. Histological examination showed that some of the envelope peptides elicited a lymphocytic cellular infiltration at the injection site, as evidenced by positive staining for CD3. Gp90 peptide-specific increases in CD3 and IFN-γ gene expression were also detected in the injection sites. Furthermore, differences were found between in vivo and in vitro responses to gp90 specific peptides. These results demonstrate a novel method for detecting in vivo cell-mediated immune responses to EIAV-specific peptides that is readily applicable to other host/pathogen systems.