The primary objectives of the present study were to characterize the antibody response elicited by infection with XMRV and to develop high-throughput antibody assays suitable for large scale epidemiologic studies of XMRV infection. Since well-characterized XMRV antibody positive human specimens and seroconversion panels are currently unavailable, the utilization of a non-human primate model of XMRV infection provides a bona fide source of positive control sera and seroconversion samples useful for assay optimization and validation.
There is a paucity of information regarding the antibody response in humans following infection with XMRV. Several studies have reported detection of relatively low levels of neutralizing antibody or antibody cross-reactive to the surrogate envelope protein of Friend Spleen Focus Forming Virus (SFFV) in patients with prostate cancer, CFS, or blood donors [5
]. Unfortunately, WB confirmation data is not available on these samples. Using recombinant based WB analysis of serum from prostate cancer patients and blood donors, Furuta et al
. detected no antibody reactivity to XMRV envelope protein but occasional reactivity to XMRV gag
]. Interpretation of these data is complicated by the lack of information regarding XMRV seroconversion patterns and suitable control reagents to determine assay sensitivity and specificity.
The present study provides the first demonstration of seroconversion patterns in primates following infection with XMRV and characterizes the nature and kinetics of the antibody response. All three experimentally infected macaques seroconverted to XMRV. The predominant antibody responses were directed against gp70, p15E and p30. Specific antibodies to gp70 and p15E appeared earlier during seroconversion and reached the highest titers. These characteristics are similar to the antibody responses elicited by MuLVs in mice [22
]. Previous studies showed that both naturally occurring and vaccine induced responses to endogenous MuLVs were predominantly antibodies against gp70 and p15E [22
]. Although antibody to p30 could be detected in certain mouse strains, the titers were lower relative to anti-gp70 or anti-p15E [23
]. In addition, a primate model to assess potential risk of retroviral-mediated gene therapy also showed similar antibody responses to amphotropic MuLV [25
]. In this study, 5 rhesus macaques experimentally infected with amphotropic MuLV developed antibodies to gp70 and p30 (anti-p15E response was not examined) that persisted through the last day tested (337-696 PI). Viral infection was also confirmed by PCR amplification of proviral DNA in peripheral blood mononuclear cells and lymph node tissue. However, the time course and titers of these specific antibody responses were not determined.
Notably, the well-characterized antibody responses in humans infected with HIV and HTLV are also primarily to the envelope proteins (HIV-gp120, HTLV-gp46), transmembrane proteins (HIV-gp41, HTLV-gp21), and core proteins of capsid and matrix (HIV-p24 and p17, HTLV-p24 and p19). Antibodies to envelope and transmembrane proteins were identified as the early and sustained serologic markers of infection [26
]. These markers are the primary targets utilized by current third generation HIV and HTLV antibody assays as well as fourth generation HIV antigen/antibody combination assays for diagnostic testing and blood donor screening [30
Taken together, the antibody responses to XMRV observed in this study are consistent with responses reported for other retroviruses. Thus, antibody responses to gp70, p15E and p30 represent potentially useful serologic markers for detection of XMRV infection.
Based on the identification of key serologic markers, prototype direct format CMIAs were developed using the recombinant proteins gp70, p15E or p30. The assays all showed good specificity (99.4-99.9%) with blood donor samples. Both gp70 and p15E prototype assays demonstrated 100% sensitivity by detecting all WB positive bleeds from XMRV-infected macaques. Seroconversion sensitivity of the p30 assay was slightly lower due to the combination of reduced analytic sensitivity and the delayed kinetics of the anti-p30 response. However, the p30 assay detects antibody to the core protein distinct from envelope proteins, thus, may still have value for confirmation of XMRV infection. Ideally, sensitivity and specificity of these prototype assays would be further validated using bona fide XMRV positive and negative human specimens once they become available.
Due to the high sequence homology, the assays described herein detect antibody responses not only to XMRV but also to other known MuLVs. Both the p15E and p30 prototype assays detected highly diluted (1:32,000 and 1:64,000) goat antibody to Friend MuLV. This is consistent with the high sequence homology of p15E (76%) and p30 (89%) proteins between XMRV and Friend MuLV. Interestingly, the gp70 assay was also able to detect the highly diluted antibodies to Friend MuLV (1:16,000) and Rauscher MuLV (1:10,000) despite lower sequence homology (59%) between the envelope proteins of these viruses. This suggests that the most conserved C-terminal domain of gp70 may represent the immunodominant region of the envelope protein.
In contrast to HIV and HTLV infection in humans that generally stimulate strong and sustained antibody responses, XMRV infection in macaques elicited detectable but less robust antibody responses. Re-infection with XMRV substantially boosted antibody titers; however, the titers decreased to a basal level by 110 days. The less vigorous antibody response may reflect a relatively low level of XMRV replication in macaques. This is consistent with the observation that only two (RIl-10 and RYh-10) of three chronically infected macaques had a detectable but low level plasma viremia (peak levels of 7,500 and ~2,000 copies/ml, respectively) after initial infection, and it was of short duration [19
]. Moreover, although all three macaques had detectable provirus in PBMC, by 30 days PI XMRV was very difficult to detect in this compartment (Figure ) Of note, XMRV was detectable in various organs and tissues throughout a 9 month follow-up using PCR, FISH and immunohistochemistry [19
]. Interestingly, a similar pattern of antibody response was also observed in HTLV-I infected pig-tailed macaques [34
]. The boosted HTLV-I antibody responses decreased to a basal level approximately 60 days following the 2nd
HTLV-I infection but remained relatively stable over the next 8 months. Thus, the relatively weak and less sustained antibody responses observed in XMRV-infected macaques may not reflect what typically occurs in humans. Further investigations are needed to determine the level and duration of antibody responses in XMRV-infected humans.