Cell mediated immunity and the prediction of CMV disease in solid organ transplant recipients

CMV is the most common viral infection after solid organ transplantation (SOT)
and is associated with significant morbidity. Without prophylaxis, most CMV
disease occurs in the first 3 months post-transplant during the period of
intense of immunosuppresion. SOT recipients at the greatest risk are those that
are seronegative recipients of organs from seropositive donors (CMV D+/R-).
Antiviral agents have proven to be useful in the prevention of CMV infection
and disease in SOT recipients, including the high-risk D+/R- patients. Upon
completion of prophylaxis however, CMV infection and disease occurs in about
50% and 25-30% respectively of D+/R- SOT recipients within the first year after
transplant. The incidence of CMV infection following prophylaxis in D+/R- lung
transplant recipients may be as high as 80%. As CMV disease occurring after
prophylaxis will continue to impact morbidity and mortality in SOT recipients,
it would be desirable to be able to predict which patient will develop this
complication. Currently, there are no reliable methods that are routinely
available to predict the risk of CMV infection or disease in an individual
patient. CMV viral load testing after prophylaxis in D+/R- patients has been
shown to have poor predictive value for subsequent CMV disease. CMV serology (a
measure of humoral immunity) was also shown to be only of marginal use in
predicting the risk of late onset disease. Cell mediated immunity (CMI) is
known to be more important than humoral immunity in controlling CMV infection.
CMV infection elicits a strong virus specific CD4+ and CD8+ T-cell response.
CD8+ T-cell responses to the virus often contain multiple antigen-specific
reactivities including to viral pp65 or IE-1 antigens as well as pp50,
glycoprotein B, and IE-2 and other antigens. CD4+ T cells also play a part in
CMV control via promotion of priming, expansion and maintenance of CD8+
CMV-specific CTLs. Measuring an individual*s CMI response to CMV may be a
useful predictor of the risk of CMV infection or disease after prophylaxis.
Patients with a poor CMI response (especially a poor CD8+ T-cell response)
could then be targeted with longer courses of antiviral prophylaxis.

Cell mediated immunity testing
Most of the previous studies have focused on CTL responses to the CMV
phosphoprotein pp65. However, since CD8+ T-cell responses to CMV often contain
multiple antigen-specific reactivities, measurement of CMI using epitopes
restricted to a single protein may not yield adequate results. In conjunction
with Dr. Rajiv Khanna (Queensland Institute of Medical Research, Australia),
and Cellestis Ltd (Sydney, Australia), we have done preliminary validation and
assessment of a CMI (Quantiferon-CMV) assay in which we measure the IFN-γ
responses to a range of T-cell epitopes of CMV viral proteins including pp65,
pp50, the glycoprotein gB, and the immediate early IE-1 antigen that are
specific for a wide range of HLA class I specificities. The assay employs a
peptide pool for stimulation of whole blood and is suitable for routine
clinical use and evaluation in multicenter studies
The Quantiferon-CMV assay has been compared to an ELISPOT assay in a study
involving 37 healthy volunteers and 25 SOT recipients. In this study, the
Quantiferon-CMV assay was at least as sensitive as the ELISPOT for some CMV
epitopes, and more sensitive for other CMV epitopes. In addition, the
Quantiferon-CMV results highly correlated with the CMV serostatus, in both
healthy volunteers and transplant recipients. In another study, the
Quantiferon-CMV assay was used in HIV-infected individuals with and without a
history of CMV disease. The CMV specific immune response measured by the
Quantiferon-CMV assay was higher in patients without a history of CMV disease,
suggesting that a positive result of the Quantiferon-CMV may predict the
likelihood for developing a protective immune response against CMV.

Preliminary Data in Solid Organ Transplantation
In our validation study in 40 healthy volunteers, IFN-γ detection had an
excellent correlation with CMV serostatus. In a single-center study done by our
group, the CMV- CMI assay was evaluated in 108 transplant recipients including
38 D+/R- patients. Detection of CMI at 3-months post-transplant had good
predictive value for protection against CMV disease (unpublished data). Based
on this study, the optimal cut-off for the test in D+/R- patients was
determined to be 0.1 IU of IFN-γ/ml.

The primary aim of this study is to determine the utility of measurement of CMV
specific cell mediated immunity using the Quantiferon-CMV assay for predicting
the risks of CMV infection and disease in D+/R- SOT recipients after the
completion of antiviral prophylaxis. It is hypothesized that those with a
strong CMI response to CMV are at low risk of subsequent CMV disease, while
those with absent or weak CMI are at high risk. Theoretically the latter group
could then be targeted for more prolonged antiviral prophylaxis.

Patients at high risk (based on pre-transplant serology D+/R-) will be followed
longitudinally to assess the development of CMV specific CMI.
CMV cell mediated immunity will be assessed at 3 time points in each patient.
1. At the time of prophylaxis discontinuation (3 or 6 months post-transplant)
2. 1-month post-prophylaxis discontinuation
3. 2-months post-prophylaxis discontinuation

In patients who develop CMV disease, a further assessment will be done at the
time of CMV disease.
The number of assessments of CMI has been kept to a minimum because 1) this
will facilitate the performance of a multi-center study, 2) this will decrease
costs, and 3) if ever used in a clinical setting, simple regimens for
evaluation of CMI will have the most direct clinical applicability.
In patients receiving 6-months of prophylaxis, testing will be performed at 6
months, 7 months and 8 months. Immunosuppression protocols will be as per the
center specific standard. Data on immunosuppression medications will be
gathered to allow for analysis of this as a potential confounder.
Laboratory methods:
Cell mediated immunity will be assessed using the Quantiferon-CMV assay. CMV
epitopes restricted through various HLA class I alleles (HLA-A1, HLA-A2,
HLA-A23, HLA-A24, HLA-B8, HLA-B35, HLA-B41, and HLA-B57) will be used in this
study. The assay is conducted in 2 parts: initially, there is an overnight
incubation of patient*s blood with the CMV synthetic peptide epitopes. The next
day, supernatant is harvested and quantification of IFN-γ production is
performed using a standard ELISA [the latter half of the assay can be performed
on frozen samples and is suitable for batch testing at a central laboratory].
All testing is performed in conjunction with a negative control (sterile
PBS/no-antigen) and a positive control (PHA, positive mitogen control). A
positive cut-point of 0.1 IU of γ-interferon will be used [this cut-point is
based on previous data from our lab studying D+/R- patients].

Possible Benefits:
It is not known whether there will be direct benefit from being in the study.
However, the information learned in this study may help other patients with
similar conditions in the future.

Possible Risks:
Taking blood is briefly uncomfortable, but not dangerous. When having blood
drawn, participant may have some bruising where it is taken. This may take
several days to go away. Every effort will be made so that blood will be
collected for the study at times when subjects are having other routine blood
tests.