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Reconstitution of Herpes Simplex Virus-Specific T Cell Immunity in HIV-Infected Patients Receiving Highly Active Antiretroviral Therapy

  1. Meghna Ramaswamy1,
  2. Anele Waters5,
  3. Colette Smith3,
  4. Emma Hainsworth1,
  5. Gareth Hardy4,
  6. Margaret Johnson2,
  7. Jonathan Ainsworth5,
  8. Andrew Phillips3 and
  9. Anna Maria Geretti1
  1. 1Department of Virology, Royal Free Hospital and Royal Free and University College Medical School, London, United Kingdom
  2. 2Department of HIV Medicine, Royal Free Hospital and Royal Free and University College Medical School, London, United Kingdom
  3. 3Department of Primary Care and Population Sciences, Royal Free and University College Medical School, London, United Kingdom
  4. 4Department of Immunology, Royal Free and University College Medical School, London, United Kingdom
  5. 5Department of HIV Medicine, North Middlesex University Hospital, London, United Kingdom
  1. Reprints or correspondence: Dr. Anna Maria Geretti, Dept. of Virology, Royal Free Hospital and Royal Free and University College Medical School, Rowland Hill St., London NW3 2PF, United Kingdom (a.geretti{at}medsch.ucl.ac.uk).

  1. Presented in part: 13th Conference on Retroviruses and Opportunistic Infections, Denver, Colorado, 5–8 February 2006 (abstract 786).

 
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Abstract

Production of herpes simplex virus (HSV)-specific interferon-γ by peripheral-blood mononuclear cells (PBMCs) of HSV-seropositive healthy donors and human immunodeficiency virus-infected persons was determined by use of ELISPOT. The mean ± SD number of spot-forming cells/106 PBMCs was 314 ± 74 in 11 healthy donors, 360 ± 69 in 3 long-term nonprogressors (LTNPs), 186 ± 52 in 9 newly diagnosed patients, and 181 ± 59 in 33 patients who were receiving highly active antiretroviral therapy (HAART) for a median period of 30 months (range, 1–109 months). In 9 patients monitored prospectively while receiving virologi-cally and immunologically successful first-line HAART, the number of spot-forming cells increased by 5.6/month (95% confidence interval, 1.2–9.9 [P=.01]) and 21.3/100 CD4 cells/mm3 gained (95% confidence interval, 13.8–28.7 [P<.0001]). Responses were correlated with LTNP status and CD4 cell count.

Herpes simplex virus (HSV)-specific T cell immunity in HIV-infected persons is impaired in comparison with that in uninfected individuals, leading to increased rates of reactivation, high levels of virus shedding, and severe mucocutaneous disease [1, 2]. The potential impact of poorly controlled HSV replication is significant. In addition to their effects on HSV disease, frequent reactivations of HSV increase the level of HIV RNA in plasma [3], with implications for HIV transmission and disease progression.

Previous studies have indicated that highly active antiretroviral therapy (HAART) has a beneficial effect on the clinical expression of HSV disease [2, 4], although HSV reactivation and shedding continue to occur frequently in treated persons [2, 5]. HAART has been associated with recovery of immune responses against the herpesviruses cytomegalovirus (CMV) and Epstein-Barr virus (EBV) [6]. Restoration of immune response may remain partial despite successful control of HIV replication [7]. Data on the rate of HAART-induced reconstitution of HSV-specific T cell immunity remain limited. In the present study, the level of HSV-specific interferon (IFN)-γ-producing CD4 T cells was measured both in HIV-infected individuals at different disease stages and in a prospective cohort that was starting first-line HAART.

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Subjects, material, and methods

The study subjects comprised 54 HSV-seropositive HIV-infected adults, 10 HSV-seronegative HIV-infected adults, 11 HSV-seropositive healthy donors, and 9 HSV-seronegative healthy donors. HSV serostatus was determined by use of the HSV IgG DiaSorin EIA kit, which does not differentiate between virus types. Demographic and clinical data were obtained from clinical databases and records. Informed consent was obtained from all subjects, and all experiments were conducted with the approval of the Ethics Committee of the Royal Free Hospital and University College Medical School, London.

In the IFN-γ ELISPOT assay, PBMCs (3×105) isolated by ficoll-hypaque density-gradient centrifugation were cultured, as described elsewhere [8], in the presence of HSV-1 (MacIntyre strain) lysate and in parallel cultures of HSV-2 (MS strain) lysate, at 3 protein concentrations (1 µg/mL, 5 µg/mL, and 10 µg/mL). Phytohemagglutinin (PHA) (5 µg/mL), uninfected Vero-cell extracts, and tissue-culture medium were used as positive and negative controls. After incubation at 37°C for 16 h, spots were enumerated by use of an automated ELISPOT reader (Karl Zeiss Imaging Associates). The number of antigen-specific IFN-γ-secreting cells was calculated as the mean of the values in triplicate wells after subtraction of background levels and was designated the “SFC value,” which was calculated as the number of spot-forming cells/106 PBMCs; a positive response was considered to be an SFC >17, which was the mean + 3 SD of the background level measured with the uninfected Vero-cell extracts and tissue-culture medium negative controls (i.e., 5 + 12). After stimulation with PHA, all samples had SFC values >400. No differences were observed between the SFC values in cultures stimulated with HSV-1 lysate and those stimulated with HSV-2 lysate, and the results are therefore described as averaged HSV-specific SFC values at a lysate concentration of 10 µg/mL. In all experiments, an antigen-dose titration was apparent; for example, in the HSV-seropositive healthy donors, at week 9 the mean ± SD HSV-specific SFC values were 67 ± 20, 128 ± 28, and 317 ± 69 at concentrations of 1, 5, and 10 µg/mL, respectively.

In the intracellular IFN-γ staining assay, PBMCs (1 × 106) were stimulated with anti-CD28 antibody (BD Biosciences) and either HSV-1 lysate and HSV-2 lysate (1 µg/mL), staphylococcal enterotoxin B (SEB) (Sigma) (1 µg/mL), or uninfected Vero-cell extracts. After 2 h at 37°C, GolgiPlug containing Brefeldin A (BD Biosciences) (1 µg/mL) was added; after an additional 14 h at 37°C, permeabilized cells were stained with labeled monoclonal antibodies (IFN-γ fluorescein isothiocyanate, CD69 phycoerythrin, CD4 peridinin-chlorophyll-protein, and CD3 allophycocyanin; BD Biosciences), were fixed with 4% formaldehyde, and then were analyzed by use of FACScan flow cytometry using CellQuest Pro Software (Beckton Dickinson).

Factors that, in unadjusted analyses, were correlated (at P<.05) with SFC values were included in a multivariable model with generalized estimating equations, to account for repeated observations. Estimates of the effect that time had on the SFC value were obtained by use of interaction terms, to account for differential changes, over time, in each patient group. All analyses were run separately for HSV-1 lysate, HSV-2 lysate, and the 2 of them averaged; no differences were observed. Statistical analyses were performed by use of SAS (version 8) software.

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Results

A total of 20 healthy donors (median age, 36 years [range, 29–78 years]) were studied prospectively; at the time when they provided samples, none were receiving anti-HSV therapy or had evidence of HSV disease. The mean ± SD HSV-specific SFC values at weeks 0, 4, and 9 were 314 ± 74, 323 ± 69, and 317 ± 69, respectively, in the 11 HSV-seropositive healthy donors and were 29 ± 8, 25 ± 10, and 30 ± 11, respectively, in the 9 HSV-seronegative healthy donors. In 4 of the HSV-seropositive donors, intracellular cytokine staining showed that HSV-specific IFN-γ-producing CD3+CD69+CD4+ T cells were present at a frequency of 1.5%–2.5%/100 HSV-specific SFC units measured by ELISPOT. No other cells stained for IFN-γ. In 1 of the HSV-seronegative donors, no IFN-γ production was detected.

In 10 HSV-seronegative patients (median age, 35 years [range, 28–54 years]) in whom the median CD4 cell count was 457 cells/mm3 (range, 264–970 cells/mm3), the mean ± SD HSV-specific SFC value was 25 ± 13. The responses of HSV-seropositive HIV-infected patients, as well as their correlation with CD4 cell counts, are summarized in figure 1.

Figure 1
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Figure 1

Production of herpes simplex virus (HSV)-specific interferon -γ, as measured by ELISPOT, in peripheral-blood mononuclear cells (PBMCs) from HSV-seropositive healthy donors and HIV-infected individuals including long-term nonprogressors (LTNPs), newly diagnosed persons, persons who had been receiving highly active antiretroviral therapy (HAART) for ≥4 weeks, and persons starting HAART. Upper panel, Responses reported as the mean ± SD SFC value, calculated as the number of spot-forming cells/106 PBMCs. Responses in LTNPs were similar (P = .58) to those in healthy donors and were significantly higher than those in newly diagnosed persons (P < .0001), persons who had been receiving HAART (P < .0001), and persons starting HAART (P = .0002). Lower panel, Relationship between HSV-specific SFC value and CD4 cell count, in all subjects; the correlation coefficient is 0.87.

The 3 HAART-naive long-term nonprogressors (LTNPs) (median age, 42 years [range, 28–45 years]) who were studied had shown CD4 cell counts >400 cells/mm3, no clinical progression, and plasma HIV-1 RNA load (PVL) levels persistently <1000 copies/mL, over a median time period of 19 years (range, 10–22 years). At the time when they provided samples, none were receiving anti-HSV therapy or had evidence of HSV disease. Responses were measured 3 times over a median time period of 33 weeks (range, 30–43 weeks). At baseline, the mean ± SD HSV-specific SFC values in this group (i.e., 360 ± 57) were similar to those in the HSV-seropositive healthy donors (P=.58) (figure 1). During follow-up, the median CD4 cell count declined from 1219 to 855 cells/mm3 (range, 370–1042 cells/mm3), and the mean ± SD SFC value declined to 310 ± 75. The mean adjusted change in SFC value was −3.6/month (95% CI, −4.8 to −2.4/month) (P<.0001).

The 9 newly diagnosed patients (median age, 35 years [range, 27–56 years]) who were studied had a median CD4 cell count of 453 cells/mm (range, 320–843 cells/mm3), and a median PVL level of 4.6 log10 copies/mL (range, 2.6–5.4 log10 copies/mL). At the time when they provided samples, 6 had evidence of HSV disease (4 cases of genital herpes and 2 cases of oropharyngeal herpes), and 3 were receiving anti-HSV therapy. The group mean ± SD HSV-specific SFC value, 186 ± 52 (figure 1) was lower than both that in the HSV-seropositive healthy donors and that in the LTNPs (P<.0001).

A total of 33 patients who were receiving HAART (median age, 40 years [range, 18–71 years]) also were studied. At the time when they provided samples, 5 of these patients had evidence of HSV disease (2 cases of genital herpes and 3 cases of oropharyngeal herpes), and 1 was receiving anti-HSV therapy. These 33 patients had been receiving HAART for a median of 30 months (range, 1–109 months), and they had been receiving their current HAART regimen for a median of 12 months (range, 1–60 months). That current regimen most frequently included ≥2 nucleoside/nucleotide reverse-transcriptase inhibitors (NRTIs) plus either a nonnucleoside reverse-transcriptase inhibitor (NNRTI) (in the case of 21 of the 33 patients) or a ritonavir-boosted protease inhibitor (PI/r) (in the case of 8 of the 33 patients). In these 33 patients, the PVL level was <50 copies/mL in 28 (84.8%) and <400 copies/mL in 30 (90.9%); the median CD4 cell count was 360 cells/mm3 (range, 90–1040 cells/mm3). The mean ± SD HSV-specific SFC value in the 13 patients receiving first-line HAART (181 ± 57) was similar to that in the 20 patients receiving other HAART treatment (182 ± 59). The mean ± SD HSV-specific SFC value (181 ± 59; figure 1) in these 33 patients was lower than that in the HSV-seropositive healthy donors and that in the LTNPs (P<.0001), was similar to that in the newly diagnosed patients (P=.74), and was marginally higher than that in the patients starting HAART (P=.08).

The final group studied comprised 9 patients (median age, 41 years [range, 30–45 years]) with a median CD4 cell count of 208 cells/mm3 (range, 5–308 cells/mm3) and a median PVL level of 4.7 log10 copies/mL (range, 3.2–5.7 log10 copies/mL) who started first-line HAART with 2 NRTIs and either 1 NNRTI (in the case of 5 patients) or 1 PI/r (in the case of 4 patients) and who achieved a PVL level of <50 copies/mL within a median time of 23 weeks (range, 7–39 weeks). At baseline, 3 of these 9 patients had HSV disease (2 cases of genital herpes and 1 case of oropharyngeal herpes), and 2 of these 3 were receiving anti-HSV therapy. During follow-up, 1 patient received suppressive anti-HSV therapy and had no clinical recurrences of HSV disease. The other patients remained untreated, and 1 of them experienced recurrent genital herpes. At baseline, the HSV-specific mean ± SD SFC value (134 ± 90; figure 1) in these 9 patients was lower than that in the HSV-seropositive healthy donors and the LTNPs (P=.0002). Over a median time period of 40 weeks (range, 26–64 weeks), the median increase in CD4 cell count was 296 cells/mm3 (range, 26–559 cells/mm3), and the median HSV-specific SFC value increased by 5.6/month (95% CI, 1.2 to 9.9) (P=.01). At the end of the follow-up period, the mean ± SD HSV-specific SFC value (183 ± 64) was still lower than that in the HSV-seropositive healthy donors and the LTNPs (P<.0001).

Comparison of all study groups showed that being either a healthy donor or an LTNP was positively correlated with higher HSV-specific SFC values. In the adjusted analysis, the mean increase in SFC value, relative to that in patients receiving HAART, was 134.8 (95% CI, 85.0 to 184.4) (P<.0001) in the healthy donors and 176 (95% CI, 92.0 to 261.0) (P<.0001) in the LTNPs. Neither sex nor age had any effect (data not shown). Comparison of HIV-infected patients showed that LTNP status and CD4 cell count were positively correlated with the HSV-specific SFC value (table 1). In patients receiving anti-HSV therapy, there was a trend toward a decreased SFC value. Overall, the HSV-specific SFC value was highly correlated with CD4 cell count (r=0.87) (figure 1) and increased by 21.3/100 CD4 cells gained (table 1).

Table 1
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Table 1

Factors associated with herpes simplex virus (HSV)-specific interferon-γ responses, as measured by use of ELISPOT, in HSV-seropositive HIV-infected individuals.

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Discussion

IFN-γ-producing CD4 T-cells play a dominant role during successful containment of herpesvirus infections, such as those caused by HSV, CMV, and EBV [9]. The present study has demonstrated that, compared with those in the healthy donors, HSV-specific IFN-γ responses are preserved in LTNPs but are significantly decreased in HIV-infected patients with progressive HSV disease. Responses recovered gradually with successful HAART and, although not directly related to the duration of therapy, were strongly correlated with an increase in CD4 cell count. Consistent with this observation are the results of intracellular cytokine staining, which showed that, in the healthy donors, HSV-specific IFN-γ production after stimulation of PBMCs with virus lysates is mediated by activated CD4 T cells.

Low-level reactivity in response to HSV stimulation was detected in the HSV-seronegative donors. This is unlikely to reflect antigenic overload but may indicate either cross-reactivity of herpesvirus-specific CD4 T cells [10] or possible priming of HSV-specific T cells during previous exposure to HSV in the absence of seroconversion [11].

LTNPs had HSV-specific IFN-γ responses comparable to those in the healthy donors, and LTNP status was found to be an independent predictor of preserved HSV-specific immunity. LTNPs are naturally able, in the absence of HAART, to control HIV viremia, without a decrease in CD4 cell count, for many years and have vigorous CD4 T cell responses; however, after several years of infection, disease progression may become apparent, so that some LTNPs may be better defined as slow progressors. This possibility is exemplified by 1 LTNP in the present study, who, during the 19 years since the initial diagnosis of HIV, had a gradual decrease in CD4 cell count, which was correlated with intermittently detectable HIV RNA in plasma. This patient lost 110 CD4 cells during 30 weeks of follow-up, and this coincided with decreasing HSV-specific IFN-γ responses. Further studies are warranted in additional LTNPs.

In the patients who started first-line HAART, increases in CD4 cell count were paralleled by a gradual recovery of HSV-specific IFN-γ responses, thereby repopulating the 0.2% of circulating CD4 T cells that, in healthy persons, contribute to the immunological memory against HSV [12]. It remains to be determined whether expansion of preexisting HSV-specific clones, de novo synthesis and selection of HSV-specific T cells, or both accounted for this recovery. The results of the present study are consistent with a previous study's finding that proliferative responses to HSV antigens improve after the initiation of HAART [13]. Nonetheless, the level of response in treated patients frequently remained below that observed in the healthy donors, despite successful virological suppression. This may explain previous studies' observations that HIV-infected persons continue to experience frequent HSV disease and high rates of HSV shedding, despite successful HAART and recovery in CD4 cell count [2, 5]. In the present study, however, clinical manifestations of HSV infection were uncommon in persons receiving HAART, indicating an overall beneficial effect on HSV disease [2, 4]. Ultimately, multiple immune and nonimmune mechanisms may be affected by HAART, which could modulate HSV disease.

HSV-specific IFN-γ responses showed a weak inverse correlation with the use of anti-HSV therapy. In earlier studies, patients treated with acyclovir had decreased T cell responses to herpesvirus antigens [14]; however, these findings have not been reproduced consistently [15]. Two explanations can be proposed. Persons with lower HSV-specific responses may have been more likely to require anti-HSV therapy than were those with better immunity. Alternatively, improved control of HSV replication may have decreased levels of HSV-specific T-cells, as a result of decreased HSV antigenic load. In line with the latter hypothesis, control of CMV viremia in HIV-infected patients has been correlated with decreased levels of IFN-γ-producing CMV-specific CD4 T cells [6].

The present study supports the conclusion that HAART enables immunological reconstitution of HSV-specific T cell responses and leads to improved control of HSV disease. The recovery appeared to be slow, despite successful HAART. We estimate that persons starting HAART who have a CD4 cell count of ∼200–250 cells/mm3 would require, on average, 33 months of immunologically successful therapy to restore HSV-specific IFN-γ responses to the levels observed in healthy donors. This provides a threshold for HSV-specific immune reconstitution that may assist with clinical management.

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Footnotes

  • Potential conflicts of interest: none reported.

  • Financial support: Royal Free and University College Medical School (London) Ph.D. training fellowship (to M.R.).

  • Received June 1, 2006.
  • Accepted September 20, 2006.
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  1. J Infect Dis. (2007) 195 (3): 410-415. doi: 10.1086/510623
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