T. A. Semenenko, E. P. Seikova, T. P. Gotvyanskaya, A. D. Gaidarenko, N. A. Polezhaeva, L. F. Evseeva, O. G. Nikolaeva
Characteristics of the immune status in specific and nonspecific prophylaxis of influenza in elderly persons
Gamaleya Research Institute of Epidemiology and Microbiology, Gabrichevsky Research Institute of Epidemiology and Microbiology, Moscow, Russia
The results of the comparative analysis of the immunological effectiveness of the anti-influenza vaccine Vaxigrip, the interferon inductor arbidol and their combination in 125 elderly persons are presented. In the process of investigation, the immunomodulating activity of the preparations under study was noted. This activity was manifested by the increase of the absolute and relative number of cells carrying CD3+, CD4+ and CD16+ markers, but not CD8+, CD19+ or CD25+; normalization of the immunoregulatory index and stimulation of phagocytic function without significant influence on the level of HLA-DR+ expression or on the concentration of the main classes of immunoglobulins. An increase in the frequency of seroconversions and the multiplicity of growth in the titers of specific antibodies to influenza viruses A (H1N1 and H3N2) and B, most pronounced in persons given arbidol along with an anti-influenza vaccine, was established.
Introduction
Despite the wide spectrum of live and inactivated vaccines developed in recent years, influenza prevention remains an urgent problem. In earlier years, the absence of 95% effectiveness with vaccine administration was attributed to either improper methods of vaccine transport or storage, or a deficiency in the immunogenic capability of the preparations used. At the present time, one of the biggest factors contributing to a patient’s failure to seroconvert, or to an insufficient period of time of antibody production, is the inability of the patient’s own immune system to adequately respond to the vaccine [8, 9].
It is well known that in the elderly, the percentage of whom increases every year in population, antiviral vaccines accomplish seroconversion much less often than in healthy young individuals. Age-related immunodeficits are associated with the shrinking of the thymus, resulting in a decrease of thymus-dependent humoral immunity and a simultaneous increase in the concentration of immunoglobulins, especially IgG and IgA, with a predominance of “less effective” antibodies [4].
In recent years, the question of using immunomodulators not only for treatment of immunodeficient conditions, but also to prevent infectious diseases, has been under discussion. The administration of immunomodulators has become more promising in the prevention of polyetiologic diseases, such as respiratory illnesses, against which effective means of prophylaxis (vaccine) has been inadequately developed. This was confirmed by T. A. Semenenko under controlled epidemiological experiments, which demonstrated immunomodulators’ effectiveness as a nonspecific preventive drug against infectious diseases [11, 12]. In addition, there is evidence of the possibility of using medications to increase the human organism’s resistance and to stimulate adequate reactivity when various vaccines are introduced into the body [3, 7, 8].
One of the preparations to show itself promisingly in recent years is the domestically produced arbidol, created in the Center for Chemical Medicines at the Chemical-Pharmaceutical Scientific Research Institute of Russia. This drug has been approved by the Russian Federation Ministry of Health for clinical use in adults[2]. Existing literature documents researchers’ interest in arbidol, which has a wide spectrum antiviral effect with RNA and DNA-containing viruses. The preventive strength of the drug against influenza and RSV has been confirmed in adult and child populations [14, 15]. These results notwithstanding, until now, there has been a lack of data on the effects of taking arbidol in conjunction with anti-influenza vaccines, which would be useful for prophylaxis during the peak of respiratory infection season. The present work undertakes the evaluation of the effect of giving arbidol along with an anti-influenza vaccine on immunity markers, and on the development of specific antigens to the influenza virus in elderly populations.
Materials and Methods
To conduct controlled epidemiological research, we used arbidol (ethyl 6 – bromo 5 – hydroxy 1 – methyl 4 – dimethyl 2 – phenylmethyl-indole 3 – carboxylate hydrochloride, monohydrate) and the split vaccine Vaxigrip (Aventis Pasteur, France), authorized for use in Russia since 1992.
The research was conducted on an elderly population (n = 125; 43 men and 82 women, median age 77.2 +/- 11.4, from 66 to 83 years), randomized in 5 groups of 25 people each. The groups received the following preparations:
Group 1: arbidol (A), 0.2 g (2 tablets) orally twice a week for three weeks
Group 2: anti-influenza vaccine (V), containing in one dose 15 mcg hemagglutinin of each virus serotype, injected subcutaneously
Group 3: arbidol 0.2 g (2 tablets) once a day orally for one week; followed by the vaccine injection
Group 4: the anti-influenza vaccine injection; then, arbidol 0.2 g (2 tablets) orally twice a week for three weeks
Group 5: placebo (control “K”)
The laboratory research was conducted both 2 weeks before, and 2 weeks after, the administration of arbidol and/or vaccine. The immunomodulator activity of the drugs was determined in accordance with methods for evaluation of immune status in humans, developed by fellows at the Ministry of Health immunology institute of the Russian Federation [10]. All participants in the experiments were followed according to the same chart. The data included: absolute and relative count of leukocytes, lymphocytes and neutrophils; cells with CD3+ phenotype (general T-lymphocytes carrying TCR-determinant); CD4+ (T-helpers); CD8+ (cytotoxic lymphocytes); CD19+ (B-lymphocytes); CD16+ (“natural killer” NK cells); CD25- (activated lymphocytes); HLA-DR+ (histocompatibility antigens); immunoregulator index (CD4+/CD8+); A, M and G immunoglobulins; circulating immune complexes and phagocyte markers, that is, cell function parameters for the humoral and phagocytic links of the immune system.
To evaluate the effects of the drugs on anti-influenza immunity markers, serological tests were done on blood serum for the presence of specific antigens of influenza groups A (H1N1 and H3N2) and B by immunoenzyme assay. To interpret the reactions we used test systems, the main components of which were respiratory virus antigens from the well surfaces of polystirol plates, and also conjugated monoclonal antibodies to human immunoglobulins (made by the Russian Academy of Medical Science and “Enterprise for the Production of Diagnostic Preparations,” St. Petersburg).
All indexes were arrived at by standard accepted statistics methods, with “Students” t-criteria, p < 0.05.
Table 1: Relative total numbers of T-lymphocytes and killer-T cells under observation in experimental and control groups
|
Groups |
CD3+ (% +/- m) |
CD4+ (% +/- m) |
||
|
I |
II |
I |
II |
|
|
1 (A) |
57 +/- 8.6 |
68 +/- 7.4**+ |
35 +/- 4.1 |
41 +/- 6.3* |
|
2 (V) |
55 +/- 5.8 |
63 +/- 8.4* |
37 +/- 8.2 |
39 +/- 7.6 |
|
3 (A + V) |
62 +/- 8.9 |
78 +/- 10.9**+ |
34 +/- 5.1 |
44 +/- 6.7**+ |
|
4 (V + A) |
58 +/- 9.6 |
67 +/- 8.6**+ |
38 +/- 8.1 |
42 +/- 7.6* |
|
5 (K) |
61 +/- 6.3 |
62 +/- 5.8 |
38 +/- 6.4 |
36 +/- 8.2 |
|
|
|
|
|
|
Notes: Here and in Tables 2 and 3: time of observation: I – before administration of the drugs; II – after drug administration; *p < 0.05; **p < 0.01 (reliability within groups); + p <0.05 (comparison of groups).
Results
Research completed before the preparations were given showed that the fundamental parameters of immunity according to mean statistical markers corresponded to a normal significance for individuals over 65 years, which confirms that the groups under study were immunologically representative of their general group.
In elderly people, the administration of arbidol and Vaxigrip provided an immunomodulating effect, leading to relatively higher numbers of T-lymphocytes and killer T-cells, which was determined with the help of monoclonal antibodies to the lymphocyte antigens (Table 1).
The relatively higher number of CD3+ and CD4+ cells was more often observed in the group which received arbidol and its combinations with the anti-influenza vaccine. Similar data resulted with regard to the absolute numbers of T-lymphocytes and killer T-cells; further, a normalization of indicators was noted especially in those who initially displayed a lower number of cells. These data are in agreement with the results of experiments by authors of other studies. The administration of the drugs under study did not lead to a significant change in absolute or relative numbers of CD8+ cells by groups as a whole, which confirms the idea that the stimulating activity of arbidol is not related to the function of cytotoxic cells.
Table 2: Phagocytic markers in participants of experimental and control groups during observation
|
Groups |
Phagocytic Marker (%) |
|
|
I |
II |
|
|
1 (A) |
46.2 +/- 4.4 |
59.2 +/- 4.1** |
|
2 (V) |
48.3 +/- 5.8 |
49.2 +/- 4.6 |
|
3 (A + V) |
50.2 +/- 4.5 |
64.3 +/- 7.6**+ |
|
4 (V + A) |
46.9 +/- 5.2 |
59.4 +/- 6.8** |
|
5 (K) |
49.2 +/- 4.2 |
49.8 +/- 5.4 |
In a large majority of elderly subjects in the study (86.4 +/- 7.6 %), we noted before the study a low immunoregulatory index (IRI, or the ratio D4+ /CD8+), which may be related to the condition of physiological immunodeficiency characteristic of advanced age. The administration of drugs led to a normalization of IRI in 52 (48.1 +/- 6.4) people, with initial lower markers, in groups 1, 3 and 4.
The important attribute of arbidol is its stimulating effect on the function of macrophages and neutrophils. In earlier experiments on animals, it was shown that the preparation not only increases the total number of macrophages with ingested bacteria, but also increases the phagocyte number [2]. A strengthening of phagocytosis was also noted in patients suffering from chronic bronchitis, which accompanied an improvement in the clinical condition of the patients and subsequently allowed some to forego antibiotics [1]. Our research confirmed the stimulating affect of arbidol, and also its effect in conjunction with the anti-influenza vaccine on the phagocytic links of the immune system, providing a heightened resilience in the patient. In the subjects of study groups 1, 3 and 4, a significant increase of phagocytic markers was seen in section II of the research (Table 3), compared to the control group.
Table 3: Absolute number of NK cells in blood serum of participants in experimental and control groups
|
Groups |
CD16+ (abc +/- m) |
|
|
I |
II |
|
|
1 (A) |
146 +/- 16.1 |
258 +/- 12.4*+ |
|
2 (V) |
158 +/- 20.1 |
163 +/- 17.5 |
|
3 (A + V) |
152 +/- 12.8 |
281 +/- 23.6**+ |
|
4 (V + A) |
143 +/- 15.3 |
248 +/- 16.7*+ |
|
5 (K) |
161 +/- 13.8 |
178 +/- 18.1 |
To all appearances, it seems that the increase in phagocytic activity in elderly people who have received arbidol and the arbidol/vaccine combination, comes from the action of cytokines – specifically, interferon “gamma.” The production of this interferon increases under the effect of arbidol.
“Natural killer” cells also function in close relation to phagocytic cells in the blood. We know that NK cells’ main function is cytolysis upon contact with cells which have been transformed or damaged by a virus (target cells). An important factor necessary for the proliferation and maturation of NK cells is IL-2, which increases the cytolytic activity of natural killer cells and widens the spectrum of target cells. NK cells, under the activation of IL-2, are transformed into LAK (lymphokine-activated killer) cells, which take direct action in the body’s defense against infections. With its strength increased under the influence of arbidol, “gamma” interferon has a direct effect on the differentiation and activation of NK cells.
Upon analysis of the markers for natural killer cell activity, we saw an increase in the NK-cell count among the experimental groups, compared with the control group (Table 3).
It appears that the stimulation of cells’ immune response, the increase in activity of NK cells, and the phagocytic process are related to an increase in the synthesis of IL-1, the phagocyte NO and the monocyte expression of HLA-DR antigens. This is shown for other immunomodulating drugs which act on the monocyte/macrophage system [6].
We noted a normalization of CD25+ and HLA-DR+ markers (in 37.6 % and 32.8 % of those under study, respectively); however, we were unable to show solid proof of the drugs’ influence on B-lymphocyte counts and the levels of basic immunoglobulin groups. With arbidol, the absence of an immunosuppressive effect in the development of specific antibodies to respiratory viruses was confirmed by immunoenzyme assay results from the experimental and control groups (before and after administration of the drugs). Moreover, comparative studies of the frequency of seroconversion with vaccine alone, versus vaccine with arbidol, allowed us to establish some distinctions between the different experimental groups.
Thus, among the elderly population who received the vaccine alone, there was an increase of the level of antibodies to virus serotype H3N2 in 53.8 %; in the antibody level to H1N1, 59.1 %; and to serotype B, 58.5% of those vaccinated. Among those who received the vaccine in combination with arbidol, the figures for an increase of antibody level to the above viruses was 64.3, 68.4 and 67.8% respectively (Group 3) and 60.0, 66.6 and 66.6% (Group 4). The seroconversion markers to influenza viruses for groups 2 and 3 are within the range established by the European Pharmacopeia for anti-influenza vaccines for ages 60 and above.
There was also an increase in specific antibody titers in the groups of subjects, on the base of combined prophylaxis, according to our various methods. The most significant rise in antibody titers was noted in those with the lowest initial levels (<20. Those who had the highest initial levels of antibodies turned out to have little change in antibody levels, when we compared serum titers from before and after the course of immunization. The mean amount by which adding arbidol to the vaccine increased specific anti-influenza antibodies was 4-6 times.
Discussion
The results of the study point to an immunomodulating effect of arbidol, of the vaccine and of the combination of the two, shown by the increase in number of T-lymphocytes and killer-T lymphocytes, the stimulation of phagocytic functions, and the induction to active state of natural killers, and also by the rise in the frequency of seroconversion and the growth in titer levels of specific anti-influenza antibodies. These results were most pronounced in Group 3, which allows us to suggest the method of giving subjects arbidol initially to be the most effective.
This compiled data is in agreement with the opinions of many experimental specialists, who believe that the administration of immunomodulators in conjunction with the anti-influenza vaccine can lead to a strengthening of the immune response, the formation of rapid endogenous defense, a hindering of hemagglutinin’s immunodepressive action, and stimulation of immune cell links with a widening of the spectrum of defense against influenza viruses for an extended time [5, 7].
The combined therapy of arbidol with the flu vaccine is promising for the over-65 population in light of its condition of physiologic immunodeficiencies. It is absolutely necessary to continue studies to determine the most successful methods and type of administration for this combined therapy of specific and nonspecific influenza prophylaxis.
Bibliography
Published 02/04/05