Adjuvant effect of low-dose interleukin-2 on antibody response to influenza virus vaccination in healthy elderly subjects

ELSEVIER

Mechanisms of Ageing and Development 77 (1994) 75-82

Adjuvant effect of low-dose interleukin-2 antibody response to influenza virus vaccination in healthy elderly subjects

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Mauro Provinciali *a, Giuseppina Di Stefano a, Mauro Colombo b. Francesco Della Croce b, Maria Carla Gandolfi b, Laura Daghetta ~', Mario Anichini c, Ruggero Della Bitta d, Nicola Fabris* "Immunology Center, INRCA Gerontology Research Department, Via Birarelli 8, 60121 Ancona, Italy bCamillo Golgi Geriatric Institute, ,4bbiategrasso, Italy cAnalysis Laboratory, INRCA Hospital, Poggwsecco, Italy dEurocetus Italia, Milan, Italy eChair of Immunology, School of Medicine, Pavia, Italy Received 3 March 1994; revision received 8 September 1994; accepted 13 September 1994

Al~rlct

It is well known that immune efficiency is frequently deteriorated in elderly people. The agediminished antibody response to T-cell dependent antigens, such as influenza virus antigens, may explain the low protection offered by influenza vaccination in the elderly population. To investigate the possibility of increasing the antibody response to influenza virus vaccinations, we have conducted a nursing home-based study on the efficacy of IL-2. Seventy-five institutionalized elderly subjects (82 + 8 years) were enrolled in the study in the course of winter season 1991-1992. Thirty-nine subjects were treated with three subcutaneous daily injections of interleukin-2 (IL-2, 1 x l06 I.U./day) before vaccination and their antibody response was compared to that of 36 aged people receiving the vaccine only. An increased antibody response against influenza virus was present in vaccine plus IL-2 treated subjects (P < 0.001) but not in subjects treated with vaccine only. The number of protected subjects 45 days after vaccination was increased only in the IL-2-treated group (P = 0.045). The low-dose of IL-2 administered and the short-term treatment allowed a good tolerance to the IL-2 injection. In conclusion, the low-dose IL-2 treatment represents an effective means of inducing antibody response to influenza virus antigens in elderly subjects without appreciable toxicity. Keywords." Interleukin-2; Antibody response; Influenza vaccine; Elderly * Corresponding author. 0047-6374/94/$07.00 © 1994 Elsevier Science Ireland Ltd. All rights reserved SSDI 0047-6374(94)01506-H

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I. Introduction

Influenza epidemics are associated with increased morbility and mortality rates in elderly people and particularly in individuals living in high-density environments, such as nursing homes [1,2]. This susceptibility to outbreaks of influenza in the elderly population has suggested they be included along with other high-risk groups for annual influenza immunization. Vaccination procedures against influenza viruses have demonstrated a limited efficacy in preventing illness among elderly nursing home subjects [3]. We and others have demonstrated the low effectiveness of standard influenza vaccination procedures in conferring adequate antibody protection in elderly subjects [2.4,5]. The main cause for the low protection offered by influenza vaccination in the elderly is certainly linked to the age-diminished antibody response particularly to the T-cell-dependent antigens, such as influenza viral antigens [61. In fact, the ageassociated thymus involution results in the reduction of T-cell-dependent functions including lymphokine production, which in turn influence the efficiency of B cells. Among lymphokines, several studies suggest an important role for interleukin-2 (IL2) on age-related decreased antibody response since IL-2 levels are reduced in the elderly population [7] and IL-2 augments B-cell growth and immunoglobulin production [8]. Recent reports have demonstrated the efficiency of IL-2 as an immunological adjuvant in several different vaccination systems by exerting protection against infections caused by virus, bacteria, and protozoa [9-13]. The recent observation of an increased production of specific antibodies against hepatitis B surface antigen in previous non-responders to vaccination, by treating them with the same vaccine combined with IL-2 [14], prompted us to explore the possibility of inducing specific production of antibodies against influenza virus in IL-2 plus vaccine treated elderly subjects and comparing them with aged people receiving vaccine only. 2. Materials and methods

2.1. Subjects and immunization procedure Seventy-five institutionalized elderly subjects aged 61-96 years were enrolled in the study in the course of winter season 1991-1992; 36 were immunized with inactivated virus vaccine alone (lsifluzonale, ISI, Italy), and 39 were immunized with inactivaled virus vaccine and interleukin-2 (IL-2, Proleukin. Eurocetus Italyl. The mean age (4- S.D.) of the subjects in each vaccine group was the same (82 + 8 years). Sixty-three subjects were female, and the gender ratio in the vaccine groups was roughly equivalent. The elderly subjects were in stable and relatively fair clinical conditions. Their principal critical clinical areas were classified as follows. Main problem: neurological (48"/;,) or psychiatric (29.3'Y,,); first associated problem: cardiovascular (16%), psychiatric (12%), neurological (10.7%); second associated problem: respiratory (4%). Subjects with cardiovascular disorders were not under therapy for congestive heart failure. Last antibiotic administration ended at least 4 weeks before enrolment. Twenty-nine subjects were not able to walk: 26 (34.7%) had cognitive. and 16 (21.3%) affective impairments: 17 (22.7%) were kept in psychic compensation

M. Provinciali et al. / Mee h

Ageing Dev. 77 (1994) 75-82

77

by psychopharmacologic therapy. Control and IL-2 treated groups did not differ in critical clinical areas, mental impairments, or pharmacologic therapy. IL-2 was administered through a subcutaneous injection in the abdominal wall at a dose of l x l06 I.U. on days -5, - 4 and -3 before vaccination (day 0). Blood withdrawal was obtained on days -5, 0, and +45 to evaluate haemanalysis, metabolic parameters and antibody titer.

2.2. Influenza antibody titration Antibodies anti viral antigens B/Yamagata 16/88, A/Taiwan 1/86 (H1NI) and A/Bejing (H3N3) were assayed by a standard haemaggiutination inhibition test [151. All serological titers were expressed as the reciprocal of the serum dilution starting from a 1:10 dilution. The anti-influenza virus antibody titers were converted to log to base 2. Antibody titers under 1:40 (5.3 log2) were considered unprotective. According to general convention, seroconversion was defined as a fourfold or greater rise in serum antibody titer when compared with the prevaccination titer.

2.3. Statistical analysis Data were analysed for statistical significance by using the two-tailed paired Student's t-test for comparing means before and after treatment, the chi-square test for comparing proportions among groups, and the McNemar's chi-square test for comparing P-values for odds ratios inside each group.

Table I Haemanalysis and metabolic signs of subjects treated with three consecutive daily injections of IL-2 Day - 5 Eritrocytes ( x 106/ram 3) Haemaglobin (g/100 ml) Haematocrit (%) Leucocytes ( × 102/mm 3) Lymphocytes ( x 102/mm 3) Neutrophils ( × 102/mm 3) Platelets ( × 103/mm 3) Glucose (mg/100 ml) Azothemia (mg/100 ml) Creatinin (mg/100 ml) SCOT (mU/ml) SGPT (mU/ml) Bilirubin (mg/100 ml) Alkaline phosphatase (mU/ml) Cholesterol (mg/100 ml) H D L cholesterol (rag/100 ml) VES (I.K.)

4.40 13.19 40.76 56.49 17.87 33.42 254.87 89.18 40.05 1.03 22.38 10.18 0.57 219.77 199.63 43.14 34.62

Day 0 ~: ~: ± ± ± :~ ± ± ± ± ± ± ~ ± ± ± ±

0.55 1.55 4.47 15.08 5.31 8.29 89.50 21.05 13.21 0.15 9.95 12.64 0.25 73.15 40.16 11.91 17.53

4.34 13.31 40.94 57.38 19.31 31.68 235.76 88.00 40.18 1.03 21.69 10.82 0.54 204.51 195.76 40.70 32.82

P-value ± ± ± :~ ± ± ± ± ± ± ± ± ± + + ± ±

0.56 1.49 4.32 16.62 4.94 9.77 71.21 25.06 15.21 0.16 10.82 11.98 0.22 60.91 39.97 10.03 17.16

NS NS NS NS 0.008 0,004 NS NS NS NS NS NS NS 0.001 NS 0.026 NS

The subjects were treated with a subcutaneous injection of IL-2 (1 x 106 I.U.) at days -5, - 4 , and - 3 before vaccination; data represent the mean ± S.D.

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M. Provinciali et al. / Mech. Ageing Dev 77 ( 1994., "5-,~'2

Table 2 Evaluation of antibody titer to influenza virus in protected and unprotected elderly subjects immunized with virus vaccine alone or virus vaccine and IL-2 Group

Day from vaccination

Antibody titer (log2) Total

Vaccine

0 45 0 45

Vagcine + 1L-2

6.21 6.44 6.08 7.19

± ± ± 4-

1.20 1.40 1.49 1.41*

Unprotected

Protected

5.16 5.37 4.59 6.30

6.29 7.16 7.12 7.80

± ± ± ±

0,36 0.62 0.73 1.30"

± ± ± ±

1.02 1.31 1.01 1.22"*

*P < 0.0001 and **P < 0.01 when compared to the respective value at day 0.

3. Results Subjects were treated with three consecutive daily injections of IL-2 at days -5 to -3 and haemanalysis, metabolic and clinical signs were evaluated before vaccination (day 0). As shown in Table 1, an increase in lymphocyte number was present after IL-2 administration (P = 0.008), while neutrophil count significantly decreased (P = 0.004). The other haemanalysis and metabolic parameters evaluated were all unaffected by IL-2 injection, apart from a slight decrease in alkaline phosphatas¢ p= 0.043

p=0.027

Treatment

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['7 Vaccine • Vaccine+ IL-2

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p= 0.026

n.s, n.s. 0 °~ e"

I

T Total population

n.s. I ~

Unprotected population

T Protected population

Fig. 1. Log of incrtmcnt of antibody liter after influenza vaccination in elderly subjects. Total population split into unprotected or protocted groups according to the antibody liter present before vaccination.

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M. Provmciali et al. / Mech. Ageing Dev. 77 (1994) 75-82

Table 3 Evaluation of the number of protected subjects 45 days after vaccination m vaccine alone or vaccine + IL-2 groups Group

Day from vaccination 0

Vaccine Vaccine + IL-2

McNemar test 45

Unprotected

Protected

Unprotected

Protected

14 14

21 22

13 5

22 31

X2

P-value

0.01 4.01

0.905 0.045

p=0.036 I - - 1

50

40

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iTreatment

30-

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Vaccine

•

Vac~t:ine + IL-2

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0 0t_ R.S.

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20

10

Total population

Unprotected population

Protected population

Fig. 2. Percentage of seroconverted subjects after influenza vaccination. Subjects immunized with vaccine alone or vaccine and IL-2 were examined for their capacity to have a fourfold or greater rise in serum antibody titer relative to the prevaccination titer.

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and HDL cholesterol levels (Table 1). As a clinical sign of IL-2 effect, a local erythema appeared in 92% of IL-2 treated subjects the day after the first injection. In 12.8% of subjects, a transient increase in body temperature over 37°C was present. No other side effects were significant, As shown in Table 2, the antibody titer against influenza virus was significantly increased in vaccine plus I L-2 treated subjects at the time ot" the second blood withdrawal (P < 0.0001), whereas no significant changes were observed in subjects receiving vaccine alone. On the basis of the antibody titer before vaccination, the subjects were divided into unprotected (antibody titer _< 1.40) or protected (antibody titer > 1.40) and their antibody response was evaluated according to the vaccine plus IL-2 or vaccine only group. As shown in Table 2, an increase in antibody titer was present in both unprotected and protected subjects treated with vaccine and IL-2 (P < 0.0001 and P < 0.001, respectively). No significant change in antibody response was observed in either unprotected or protected subjects who received the vaccine alone. The analysis of antibody titer as a mean of log of increment for each subject revealed a significant enhancement in antibody response in vaccine plus IL-2 treated subjects (P = 0.027) but not in subjects treated with vaccine alone (Fig. 1). Both unprotected and protected subjects treated with vaccine and IL-2 reported a significant log increase of antibody titer particularly evident in unprotected subjects (P = 0.043 and P = 0.026, respectively). The number of protected subjects was significantly higher 45 days after vaccination than before vaccination in vaccine plus IL-2 treated group (P = 0.045), but not in vaccine only treated subjects (P = 0.905) (Table 3). As reported in Fig. 2, seroconversion frequencies (percentage of subjects seroconverted) were significantly higher in vaccine plus IL-2 subjects in comparison to vaccine only treated subjects, considering either total or unprotected populations (P = 0.016 and P = 0.036, respectively). The percentage of seroconverted was also higher in vaccine plus IL-2 than in vaccine alone protected subjects (Fig. 2) though not to a statistically significant level. 4. Discussion

The well studied role of IL-2 in the immune response has suggested the possibility that IL-2 administrations might be used to boost immune responses when these are inadequate. Previous observations have demonstrated the usefulness of IL-2 in experimental infectious diseases as shown by the protection exerted by in vivo IL-2 administration against viruses, such as Herpes Simplex, various bacteria, and protozoa [9-13]. I L-2 has also been shown to overcome genetic unresponsiveness to certain vaccine antigens and to enhance the immune response to viral vaccine in immunocompromised mice [ 16-19]. Furthermore, IL-2 added to conventional vaccines has been shown to enhance resistance to later challenge [11]. In human infectious diseases, IL-2 was shown to have a high efficacy without significant toxicity [14]. Our recent study, in agreement with other surveys on influenza vaccination in old age, has reported the poor effectiveness of standard vaccination procedures in elderly people [2,4,5].

M. Provmciali et al./Mech. Ageing Dev 77 ~1994) 75-62

81

The existence of a relationship between decreased IL-2 production, reduced antibody response and low effectiveness of influenza vaccination in elderly subjects has indicated the possibility of ameliorating the immune response in aged people by IL-2 administration. The results reported in this study demonstrate for the first time the possibility of enhancing the antibody response to influenza virus through IL-2 treatment in institutionalized elderly subjects. The IL-2 recipients not only had higher antibody titers, but, more relevant, nine of 14 unprotected recipients of vaccine plus IL-2 developed protective antibody titers, as compared to only one of 14 vaccine alone recipients (Table 2). The higher seroconversion frequencies observed in unprotected vaccine plus IL-2 subjects in comparison to the protected group (50.00 vs. 18.18%, respectively) suggest that IL-2 mainly acts on the elderly population having lower basal antibody titer, i.e. on the more immunocompromised population. With regard to the mechanism by which IL-2 induces a higher antibody response to influenza vaccine, our supposition is that the low-dose IL-2 administration supplies the reduced synthesis of IL-2 present in human aging [7] and, consequently, leads to a better B cell proliferation and differentiation to immunoglobulin-secreting cells [8]. In conclusion, our results clearly show that the use of immunomodulating substances, such as IL-2, may recover the age-related-defect in immune reactivity present in elderly subjects. The low-dose of IL-2 administered and the short-term treatment allowed a good tolerance to IL-2 injection without appreciable toxicity. Our findings demonstrate that low-dose IL-2 treatment represents an effective means of inducing systemic antigen-specific immune response in elderly subjects and offers a new approach to prevent age-related infectious diseases.

Acknowledgements This work was supported by the Italian Health Ministry targeted project: 'Potenziamento della vacciiaazione antiinfluenzale nell'anziano'. The authors thank Mrs B. Bartozzi, Mr A. Manzotti and Mr M. Marcellini for their technical assistance.

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