Several epidemiological studies of older individuals have demonstrated an association between an elevated CMV-specific immune response and a relative reduction in overall survival. As such, an understanding of the immunological mechanisms that drive memory inflation is important for the development of potential therapeutic interventions that may enhance healthy aging.
CMV-specific memory inflation is seen in both the cellular and humoral arms of the immune response, but there is debate as to the importance of viral antigen in driving this process. The M38, m139, and IE3 epitopes that undergo the most profound inflation exhibit a differentiated effector phenotype and show impaired production of interleukin-2 (IL-2), both of which reflect a history of repeated antigen stimulations (13
). As such, the presence of a peptide antigen might be expected to play an important role. Previous attempts to suppress herpesvirus-specific T-cell immunity using antiviral drugs have shown limited success. A model of systemic murine HSV infection has been used to generate an immune profile that resembles memory inflation, and antiviral therapy suppressed immunity by approximately 30% following 7 months of therapy when it was started soon after infection (36
). The role of antigen dependence has also been tested in MCMV models in which antivirals were administered to animals infected with a famciclovir-sensitive MCMV mutant strain, and suppression of cellular immunity was not evident after 12 weeks of treatment (27
). We chose to use valaciclovir in our studies, as it has demonstrated excellent efficacy against MCMV in vitro
and is also well tolerated in human subjects for periods of prolonged treatment. We did not select a more potent inhibitor, such as ganciclovir, as valaciclovir has markedly fewer side effects and does not have further toxic bystander effects on hematopoietic progenitor cells (37
Memory inflation in humans is seen most markedly in elderly individuals, and we therefore chose to study mice aged 18 months in our studies. There has been very little previous analysis of this group, possibly due to the technical and financial requirements of maintaining virally infected animals for prolonged periods of time. Recently, den Braber et al. (2012) showed that naïve T-cell frequencies in elderly mice are almost exclusively sustained by thymic output, whereas the naïve T-cell pool of humans in this age group relies largely on homeostatic proliferation (40
). Therefore, it might be anticipated that the effect of antiviral treatment is less pronounced in CMV-infected humans than in mice, although in this regard, it is of interest that MCMV-specific memory inflation does still occur in thymectomized mice (41
). It has been suggested that both the TN
pools proliferate in response to antigen stimulation to maintain CD8+
T-cell memory inflation (42
); therefore, future studies should consider how age and the extent of thymic output may contribute to inflationary processes.
Sophisticated adoptive transfer studies of MCMV-specific T cells into naïve and infected mice have estimated that the half-life of inflationary MCMV-specific T cells is around 45 to 60 days (14
). However, we found that 3 months of antiviral treatment was insufficient for reducing T-cell inflation, with reductions in peptide-specific immunity starting to become evident after 6 months and profound suppression of the MCMV CD8+
T-cell response after 12 months across a range of both the inflationary and noninflationary MCMV peptides. Furthermore, treatment led to an almost 80% reduction in the combined CD8+
T-cell response to five MCMV-immunodominant CD8+
T-cell epitopes in elderly mice. It should be noted that the mice had been infected for at least 6 months prior to starting antiviral therapy, so the model therefore begins to resemble the situation in the human population, where individuals will have been infected for many years prior to potential consideration of antiviral therapy. In addition, the MCMV-specific CD8+
T-cell responses were not completely abrogated, certain responses (e.g., m139) were not affected by antiviral treatment, and factors such as homeostatic proliferation are likely to retain a residual role. An interesting feature that we observed was that the degree of differentiation of the remaining population of the IE3-specific lymphocytes was markedly reduced (), indicating that reactivation is required to both promote the differentiation and maintain the majority of the MCMV-specific CD8+
It was of interest to measure the rate of decay of the MCMV-specific immune response following the introduction of antiviral therapy. The half-lives for the five immunodominant MCMV-specific CD8+
T-cell populations were between 200 and 250 days when taken over the 12-month period (). This is considerably longer than has previously been estimated for MCMV-specific responses (14
), although it was of interest to note that the kinetics of T-cell decline were not linear. There was a tendency for the t1/2
of decay to shorten with time; the IE3-specific response was the most marked in this regard, with cells declining to a t1/2
of 45 days in the last 6 months of treatment, which we hypothesize was due to a more complete suppression of the IE3 antigen.
Factors that may influence the degree of memory inflation are the nature and origin of the target antigen. The levels of suppression of CD8+
T-cell immunity against individual peptide epitopes were comparable in our study. However, the most profound reduction was seen in the response to IE3, which is a highly inflationary epitope and has demonstrated some unique biological features in the C57BL/6 model. In particular, the response develops late after infection and is highly dependent on the presence of a CD4+
T-cell response (43
). It has been suggested that priming of the IE3-specific immune response may require CD4-mediated licensing of dendritic cells (43
). As such, it is possible that the uptake of viral protein by dendritic cells is suppressed during valaciclovir treatment and leads to the dramatic suppression of this response. Snyder et al. (2011) have also shown that IE3-specific CD8+
T cells do not accumulate if famciclovir is administered both prior to and immediately following MCMV infection (27
). We also observed partial decreases in the frequency of M45- and M57-specific lymphocytes during the most prolonged course of treatment. These reductions were statistically significant for only M45; as such, we believe that the classification of M45 and M57 as noninflationary populations of MCMV-specific T cells may not be definitive, as these cells were also lost following adoptive transfer (14
), indicating some requirement for antigen stimulation.
Our study has shown that valaciclovir needs to be taken for prolonged periods in order to suppress the accumulation of CMV-specific T cells, and this raises the question of the potential toxicity of long-term administration of the drug. Indeed, it has been reported that aciclovir can cause apoptosis of murine CD8+
T lymphocytes at high doses in vitro
and in vivo
). Here, we used the antiviral drug at half the dose others have used and did not observe any reductions in the actual numbers of viable cells from the spleen (). In addition, if valaciclovir was lymphotoxic specifically toward CD8+
T cells, one would expect an increase in the CD4/CD8 T-cell ratio; however, this was not the case, and MCMV+AV-treated mice had CD4/CD8 T-cell ratios that were equivalent to those of MCMV-neg mice (data not shown). It is notable that the drug has been in clinical use for over 20 years with no reports of aciclovir-induced lymphopenia. The loss of MCMV-specific cells is therefore unlikely to be a result of direct CD8+
T-cell lymphotoxicity, and this is reinforced by the observation that the magnitude of NP-specific CD8+
lymphocytes was not affected by antiviral treatment.
Two features of CMV infection in humans are that it markedly increases the number of memory cells in the peripheral blood and reduces the naïve T pool. These factors could impair immune function in CMV-seropositive donors, and it is important to observe that antiviral treatment is able to correct this effect in the murine model. Naïve T cells comprised 37% of CD8+ T cells in the 18-month-old MCMV-neg mice, but this was reduced to only 15% in the MCMV-infected group. Remarkably, valaciclovir treatment was able to completely reverse this to a value of 42%. The effector memory CD8+ T-cell pool was almost doubled in frequency in the virally infected group, and this increase was completely suppressed with antiviral treatment. These observations seem particularly auspicious for a potential therapeutic role of valaciclovir in reversing the expansion of clonal CMV-specific CD8+ T cells within the peripheral blood of CMV-infected adult humans.
The use of an antiviral agent to suppress the CMV-specific immune response will only be of value if it improves the functional immune capacity of the host. In order to assess this, we used influenza as a pathogen challenge in our study groups. This is a relevant model, as influenza infection remains a major cause of death in elderly humans, and CMV-specific immunity has been shown to have been an important determinant of vaccine-dependent immunity in some studies (31
). It was of interest that the most severe clinical features following IAV infection were seen in the MCMV−AV group, and antiviral therapy was able to prevent these. The magnitude of the IAV-specific CD8+
immune response was comparable in all the study groups, but the degree of T-cell differentiation was suppressed in the antiviral group, suggesting a potential role of chronic MCMV infection in driving T-cell differentiation against heterologous pathogens. Indeed, this has been observed in relation to the CD4+
T-cell response in CMV-positive human subjects (45
). One additional and intriguing observation was that some clinical and immunological differences following the IAV challenge were seen between valaciclovir-treated animals and MCMV-neg mice. MCMV infection has been shown to have beneficial effects against an unrelated pathogen challenge in young mice (46
), and it is possible that antiviral treatment may unmask a potential benefit of an underlying MCMV infection that is otherwise lost in elderly animals due to uncontrolled memory inflation.
The data presented in this article have important implications for the potential treatment of patients with high levels of CMV-specific immunity that have been associated with immune senescence (47
). Using the MCMV model, we have shown that memory inflation is reversible and is dependent on viral replication. Unfortunately, we were unable to show that antiviral treatment reduces the viral load, as MCMV titers were undetectable in latently infected mice. Valaciclovir is often given for periods of many years in order to suppress the reactivation of genital herpesvirus infection, and it is well tolerated. Viral resistance to valaciclovir is rare and is most commonly seen in the setting of immune suppression. The amount of valaciclovir used in our study was equivalent to the dose administered for the treatment of acute varicella zoster infection or control of CMV replication in patients undergoing immune suppression. However, the dose of valaciclovir that would be required to suppress CMV replication in immunocompetent subjects may be much less than this and is currently the subject of investigation. It may now be appropriate to consider the use of valaciclovir in CMV-seropositive people to determine if this can offer novel clinical opportunities to improve the health of the elderly population.