Antiretroviral drug resistance, drug toxicity and adherence are major concerns in clinical management of HIV infection [1Pokrovskii VV. Treatment of HIV-infections: success or crisis? Ter Arkh 2001; 73: 52-4.]. The combination of antiviral drugs, such as highly active antiretroviral therapy (HAART), may prevent HIV from mutating and spreading, allowing patients to rebuild their immune system to the same levels as in normal individuals [2Mocroft A, Phillips AN, Gatell J, et al. EuroSIDA study group. Normalisation of CD4 counts in patients with HIV-1 infection and maximum virological suppression who are taking combination antiretroviral therapy: an observational cohort study Lancet 2007; 370: 407-13.]. On the other hand, the immune activation caused by enhanced immune reaction to HIV is now recognized as a cause of depletion of CD4 T-cells and resulting immunodeficiency [3Appay V, Boutboul F, Autran B. The HIV infection and immune activation: "to fight and burn" Curr Infect Dis Rep 2005; 7: 473-9.]. These problems are a driving force for research on new therapies that can provide suitable solutions. The most optimal therapeutic solution is an effective and safe immunotherapy that could regulate the immune response in a manner favorable to the host. There are many types of immune modulators that have been used clinically for viral infections, but for HIV the choice of immune interventions is limited [4Ershov FI. Use of immunomodulators in viral infections Antibiot Khimioter 2003; 48: 27-32.].

Ukraine has the highest prevalence of HIV infection in Eastern Europe [5Kelly JA, Amirkhanian YA. The newest epidemic: a review of HIV/AIDS in Central and Eastern Europe Int J STD AIDS 2003; 14: 361-71., 6van der Werf MJ, Yegorova OB, Chentsova N, et al. Tuberculosis-HIV co-infection in Kiev City, Ukraine Emerg Infect Dis 2006; 12: 766-8.]. Oral immunomodulator Dzherelo is used in Ukraine for the management of HIV infections, including patients co-infected with TB [7Chkhetiany R, Pylipchuk V, Argzanova O, et al. Comparative effect of an immunomodulator Immunoxel (Dzherelo) when used alone or in combination with antiretroviral therapy in drug-naïve HIV infected individuals Int J Biotechnol 2007; 9: 267-76.-12Zaitseva SI. Clinical efficacy of phytopreparation Dzherelo and its influence on the functional status of liver in patients with destructive forms of tuberculosis Probl Ecol Med Gen Clin Immunol 2006; 71-72: 132-40.]. Clinical studies have indicated that Dzherelo can significantly increase CD3 and CD4 T-lymphocyte populations and helps to achieve better clinical response when combined with standard anti-retroviral therapy (ART) consisting of zidovudine (AZT); lamivudine (3TC), and efavirenz (EFV) [7Chkhetiany R, Pylipchuk V, Argzanova O, et al. Comparative effect of an immunomodulator Immunoxel (Dzherelo) when used alone or in combination with antiretroviral therapy in drug-naïve HIV infected individuals Int J Biotechnol 2007; 9: 267-76.-9Prihoda ND, Arjanova OV, Yurchenko LV, et al. Adjuvant immunotherapy of tuberculosis in drug-resistant TB and TB/HIV co-infected patients Int J Biomed Pharm Sci 2008; 2: 59-64.]. Dzherelo has been found to decrease the incidence of opportunistic infections and reverse AIDS-associated wasting [7Chkhetiany R, Pylipchuk V, Argzanova O, et al. Comparative effect of an immunomodulator Immunoxel (Dzherelo) when used alone or in combination with antiretroviral therapy in drug-naïve HIV infected individuals Int J Biotechnol 2007; 9: 267-76.-9Prihoda ND, Arjanova OV, Yurchenko LV, et al. Adjuvant immunotherapy of tuberculosis in drug-resistant TB and TB/HIV co-infected patients Int J Biomed Pharm Sci 2008; 2: 59-64.]. Dzherelo has also been found to decrease the hepatotoxicity associated with ART [7Chkhetiany R, Pylipchuk V, Argzanova O, et al. Comparative effect of an immunomodulator Immunoxel (Dzherelo) when used alone or in combination with antiretroviral therapy in drug-naïve HIV infected individuals Int J Biotechnol 2007; 9: 267-76.-9Prihoda ND, Arjanova OV, Yurchenko LV, et al. Adjuvant immunotherapy of tuberculosis in drug-resistant TB and TB/HIV co-infected patients Int J Biomed Pharm Sci 2008; 2: 59-64., 12Zaitseva SI. Clinical efficacy of phytopreparation Dzherelo and its influence on the functional status of liver in patients with destructive forms of tuberculosis Probl Ecol Med Gen Clin Immunol 2006; 71-72: 132-40.].

Dzherelo was approved in 1997 by the Ministry of Health of Ukraine as an immunomodulating supplement, which so far has been used by over 500,000 individuals for various indications including chronic bacterial and viral infections such as TB and HIV, autoimmune diseases, and malignancy [7Chkhetiany R, Pylipchuk V, Argzanova O, et al. Comparative effect of an immunomodulator Immunoxel (Dzherelo) when used alone or in combination with antiretroviral therapy in drug-naïve HIV infected individuals Int J Biotechnol 2007; 9: 267-76.]. Dzherelo contains concentrated aqueous-alcohol extract from medicinal plants such as Aloe, Common knotgrass, Yarrow, Purple coneflower, Tutsan, Centaury, Snowball tree berries, Nettle, Dandelion, Sweet-sedge, Oregano, Marigold, Seabuckthorn fruit, Elecampane, Tormentil, Greater plantain, Wormwood, Siberian golden root, Cottonweed, Licorice, Fennel, Birch tree fungus, Thyme, Three-lobe Beggarticks, Sage, Dog rose fruit, and Juniper fruit. Our study was aimed at evaluating the effect of Dzherelo on immune cell subsets and viral load among HIV patients treated with standard ART in comparison to a control population which received ART alone.

After one month on the therapy there was a clear distinction between recipients of ART alone and those who received ART along with the daily dose of Dzherelo. This disparity became even more evident at the end of 2^nd month of therapy. Some but not all immune markers of lymphocyte subsets were impacted in a statistically significant manner. The changes in viral load among HIV patients of both groups also reached statistical significance. These findings are described in detail below.

CD3+ Total T Lymphocytes

After one month on ART alone the absolute and percent (%) values of total CD3+ lymphocytes per microliter of blood changed in a statistically discordant manner i.e. from a baseline 664(36.5%) to 743(38.4%) with P=0.13(P=0.03), as analyzed by paired Student t-test. Similarly, after one month in the group receiving Dzherelo there was a statistical discordance between absolute and percent CD3+ values: from 595(34.2%) to 664(40.5%); P=0.14(P=0.0003). After 2 months the number of total CD3+ lymphocytes increased further to 785 (43.9%) in arm B (Dzherelo) i.e. P=0.034 (P=0.0002), whereas in the control it increased to 819 (39.8%) cells, with probability values P=0.06 (P=0.03) respectively. The accrual in total lymphocytes from baseline to the end of follow-up, was 23.3% and 31.9% for absolute and 9% and 28.4% for relative numbers in control and Dzherelo arms respectively (Fig. 1).

Fig. (1) Changes in absolute and relative numbers of peripheral blood cell subpopulations at 2 months post-therapy as expressed in percentage values (Y-axis) relative to their respective baseline levels.

Viral Load

The viral load, as measured by plasma RNA-PCR at baseline and at the end of 2^nd month, decreased in the ART group (1718 to 1419 copies/ml; P=0.008), as analyzed by Wilcoxon signed rank test. In the Dzherelo arm the viral load decreased from 1793 to 1368 copies; P=0.001). About three-quarters (14/19) of patients on ART alone displayed the decrease in viral load, while 18 out of 19 of patients on Dzherelo (95%) had a reduction in their number of viral copies (Table 1).

Table 1.

Effect of 2-Month ART without or with Dzherelo on HIV-RNA Plasma Levels

In prior studies Dzherelo has been shown to influence positively CD3 and CD4 lymphocyte numbers [7Chkhetiany R, Pylipchuk V, Argzanova O, et al. Comparative effect of an immunomodulator Immunoxel (Dzherelo) when used alone or in combination with antiretroviral therapy in drug-naïve HIV infected individuals Int J Biotechnol 2007; 9: 267-76.-9Prihoda ND, Arjanova OV, Yurchenko LV, et al. Adjuvant immunotherapy of tuberculosis in drug-resistant TB and TB/HIV co-infected patients Int J Biomed Pharm Sci 2008; 2: 59-64.]. Dzherelo reduced the incidence of opportunistic infections and reversed body weight loss associated with HIV [7Chkhetiany R, Pylipchuk V, Argzanova O, et al. Comparative effect of an immunomodulator Immunoxel (Dzherelo) when used alone or in combination with antiretroviral therapy in drug-naïve HIV infected individuals Int J Biotechnol 2007; 9: 267-76., 10Nikolaeva LG, Maystat TV, Pylypchuk VS, Volyanskii YuL, Masyuk LA, Kutsyna GA. Effect of oral immunomodulator Dzherelo (Immunoxel) in TB/HIV co-infected patients receiving anti-tuberculosis therapy under DOTS Int Immunopharmacol 2008; 8: 845-51.]. It has also been shown to reduce the toxic side effects of ART, hepatotoxicity in particular [7Chkhetiany R, Pylipchuk V, Argzanova O, et al. Comparative effect of an immunomodulator Immunoxel (Dzherelo) when used alone or in combination with antiretroviral therapy in drug-naïve HIV infected individuals Int J Biotechnol 2007; 9: 267-76., 9Prihoda ND, Arjanova OV, Yurchenko LV, et al. Adjuvant immunotherapy of tuberculosis in drug-resistant TB and TB/HIV co-infected patients Int J Biomed Pharm Sci 2008; 2: 59-64., 12Zaitseva SI. Clinical efficacy of phytopreparation Dzherelo and its influence on the functional status of liver in patients with destructive forms of tuberculosis Probl Ecol Med Gen Clin Immunol 2006; 71-72: 132-40.]. For example, elevated liver aminopeptidase ALT and AST levels caused by ART have been shown to return back to normal levels when Dzherelo is used. However, these studies have not dealt with the effect of Dzherelo on other immune markers and viral load.

Our 2-month study conducted in prior anti-retroviral drug-naïve population reveals that when Dzherelo is added to ART there are significant benefits associated with this intervention. In our hands Dzherelo appears to display the same effect as reported by independent investigators. Our results indicate that administration of Dzherelo along with ART can produce significant increase in total CD3+ lymphocytes, CD4+ helper cells, better CD4/CD8 ratio, higher number of CD3+HLA-DR+ activated lymphocytes, and NK cells. Dzherelo appears to increase absolute but not relative numbers of CD8+ T lymphocytes and reduce significantly CD20+ B lymphocyte subpopulation. Furthermore, Dzherelo appears to contribute to the inhibitory effect of ART on viral replication resulting in statistically significant lower viral load in a higher proportion of patients (Table 1). These effects could not be attributed to heterogeneity of patient populations in two tightly matched treatment groups in which none of the initial characteristics in terms of age, gender, disease stage and cell counts were statistically different at baseline.

It is well established that elevated CD3 and CD4 counts and higher CD4/CD8 ratio are associated with better prognosis in patients with HIV [13Bogner JR, Goebel FD. Lymphocyte subsets as surrogate markers in antiretroviral therapy Infection 1991; 19: S103-8.]. For this reason Dzherelo is likely to influence positively the outcome of treatment and disease progression in our study population. Similarly, viral load is a predictor of HIV disease progression, its persistent elevation in HIV infected patients being indicative of poor prognosis [14Arduino JM, Fischl MA, Stanley K, Collier AC, Spiegelman D. Do HIV type 1 RNA levels provide additional prognostic value to CD4(+) T lymphocyte counts in patients with advanced HIV type 1 infection? AIDS Res Hum Retroviruses 2001; 17: 1099-5., 15Dybul M, Fauci AS, Bartlett JG, Kaplan JE, Pau AK. Panel on clinical practices for treatment of HIV. Guidelines for using antiretroviral agents among HIV-infected adults and adolescents Ann Intern Med 2002; 137: 381-433.]. While there were earlier indications that Dzherelo may reduce the viral burden, our study is the first to report this phenomenon in a systematic fashion. Despite the fact that the HIV RNA levels had decreased by less than a log the difference between baseline and outcome levels was highly significant (Table 1). While baseline and outcome viral burden appeared unusually low, this may have been due to the PCR kit we have used in this study; since we have seen similar low numbers in our study dealing with TB/HIV dually infected patients [10Nikolaeva LG, Maystat TV, Pylypchuk VS, Volyanskii YuL, Masyuk LA, Kutsyna GA. Effect of oral immunomodulator Dzherelo (Immunoxel) in TB/HIV co-infected patients receiving anti-tuberculosis therapy under DOTS Int Immunopharmacol 2008; 8: 845-51.]. Nevertheless, it is likely that the observed effect on viral load is mediated by immune cells since Dzherelo does not have a direct effect on HIV replication [7Chkhetiany R, Pylipchuk V, Argzanova O, et al. Comparative effect of an immunomodulator Immunoxel (Dzherelo) when used alone or in combination with antiretroviral therapy in drug-naïve HIV infected individuals Int J Biotechnol 2007; 9: 267-76.].

Our study reveals that at the end of study the patients on Dzherelo had absolute and relative numbers of CD3+HLA-DR+ cells equal to 348 (36.5%) or 216% and 40.9% more compared to baseline, whereas patients on ART had their numbers increased to a maximum 26.3%. Compared to normal blood donors the relative numbers in Dzherelo group were comparable but absolute values were lower i.e. 35.2% and 585 respectively. It has been shown that HIV+ patients usually have significantly higher HLA-DR expressing cells, which may negatively affect T cell immune responses and facilitate the disease progression [16Mahalingam M, Peakman M, Davies ET, Pozniak A, McManus TJ, Vergani D. T cell activation and disease severity in HIV infection Clin Exp Immunol 1993; 93: 337-43.]. Others suggested that the increase in HLA-DR-reactive T lymphocytes was not predictive of clinical outcome [17Levacher M, Tallet S, Dazza MC, Dournon E, Rouveix B, Pocidalo JJ. Comparison with CD4+ lymphocyte count in non-progressors and progressors towards AIDS Clin Exp Immunol 1990; 81: 177-82.]. We do not know to which subpopulation of T lymphocytes the CD3+HLA-DR+ activated cells belong. Judging from relative changes in CD4 and CD8 subsets it is possible that the activation marker is expressed on CD4 cells. However, due to limitations of the commercially available panel of antibodies preventing simultaneous staining with multiple fluorescent markers, we have not been able to verify this possibility. This needs to be ascertained in future studies.

Although a great deal of information is available about the role of T lymphocytes in the immune response against HIV comparatively little is understood regarding the involvement of B lymphocytes. Our results show very little variation in CD20+ B-cell numbers in the ART-treated arm but a highly significant decrease in Dzherelo recipients to levels that were identical to those seen in normal individuals i.e. 333 (23.3%) vs 337 (22.2%). Contrarily, the absolute and relative numbers of B-cells were more than double in ART recipients. We do not know what the significance of this observation is. It has been reported that the expression of CD20 is consistently higher when B cells from HIV-infected individuals are compared with those from uninfected subjects [18Ginaldi L, De Martinis M, D'Ostilio A, Marini L, Quaglino D. Changes in antigen expression on B lymphocytes during HIV infection Pathobiology 1998; 66: 17-23.]. Thus, we can only assert that Dzherelo is highly effective in restoring the normal balance of CD20 expressing B-cells.

We have observed the drastic difference between two arms in relative number of NK cells after 2 months of therapy. When compared to the entry levels the patients on Dzherelo had 52.7% more of NK cells whereas the population of these cells had increased by only 15.5% in ART alone group. The role of NK cells in HIV remains unclear. While some indicated that higher number of NK cells is critical for controlling HIV, others indicated that the quality but not the number of cells is critical in defending the host against HIV infection [19Bruunsgaard H, Pedersen C, Skinhoj P, Pedersen BK. Clinical progression of HIV infection: role of NK cells Scand J Immunol 1997; 46: 91-5., 20Alter G, Malenfant JM, Delabre RM, et al. Increased natural killer cell activity in viremic HIV-1 infection J Immunol 2004; 173: 5305-11.]. This discrepancy probably stems from differences in the design of studies some of which were based on measurement of functional activity but others were based on the enumeration of cells at various stages of the disease. Since we have not measured the functional activity of NK cells we do not know what is the significance of this phenomenon in regard to the immunopathogenesis of HIV. Based on the evidence that Dzherelo is highly effective as an immune adjuvant to ART we can only speculate that in our case the increase in NK numbers might be beneficial to the host.

Many studies have been conducted aimed at determining the phenotype of immune cells in HIV infection. While there is a consensus that the immune response plays a critical role in determining the clinical outcome much more has to be learned in order to have a clear picture of cellular events during the course of disease. The understanding of the immune mechanism controlling HIV may result in design of better vaccines and immunotherapies. Therefore, high priority should be given to efforts of prevention and treatment of infectious complications in these patients.

Currently available chemotherapy for the treatment of HIV is not ideal, requiring multiple drugs to be taken in combination for the rest of life of a patient [1Pokrovskii VV. Treatment of HIV-infections: success or crisis? Ter Arkh 2001; 73: 52-4.]. The extended duration of therapy, coupled with the side effects, often results in poor patient adherence, treatment failure, and the emergence of drug resistance with major social and economic implications. We believe that immunotherapy is an indispensable part of therapeutic strategies against HIV. The development of novel immune-based therapies is an urgent objective for anti-HIV drug discovery. Many immune interventions are available against bacteria, protozoa, fungi and viruses. While often effective the mechanism of many immunomodulators is poorly understood. This downside should be balanced against clinically confirmed benefits. Our study provides an early glimpse into the putative immune mechanism of Dzherelo, which has been successfully used as an immune adjunct to HIV therapy in Ukraine during last ten years [7Chkhetiany R, Pylipchuk V, Argzanova O, et al. Comparative effect of an immunomodulator Immunoxel (Dzherelo) when used alone or in combination with antiretroviral therapy in drug-naïve HIV infected individuals Int J Biotechnol 2007; 9: 267-76.-12Zaitseva SI. Clinical efficacy of phytopreparation Dzherelo and its influence on the functional status of liver in patients with destructive forms of tuberculosis Probl Ecol Med Gen Clin Immunol 2006; 71-72: 132-40.]. Additional studies are needed to develop better understanding of Dzherelo’s properties and to supplement the current arsenal of HIV therapies.