GRANULOCYTE MACROPHAGE COLONY STIMULATING FACTOR (GM-CSF) INDUCED SERO-PROTECTION IN END STAGE RENAL FAILURE PATIENTS TO HEPATITIS B IN VACCINE NON-RESPONDERS

Abstract

Hepatitis B (HB) virus infection is a major health problem in dialysis dependent end stage renal failure (ESRF) patients. The sero-conversion rate after recombinant HB vaccine in ESRF patients is poor. Adjuvants like Granulocyte Macrophage-Colony Stimulating Factor (GM-CSF) have been found to improve response rate to vaccines. This study was conducted to evaluate the efficacy of GM-CSF as an adjuvant to HB vaccine in ESRF patients who were non-responders to the usual three double dose vaccinations (primary non-responders). Fifty consecutive HBsAg negative and anti-HBs negative ESRF patients on hemodialysis over thirty months were prospectively included (Jan. 96-June 98). All received 40 ug of recombinant HB vaccine at 0, 1, 2 month interval. Anti-HBs titres were subsequently tested after four weeks of the third dose. There were 19 (38%) primary non-responders (antiHBs negative). Twelve (Group I) of primary non-responders were given an additional dose of HB vaccine with 300 ug (5–6 ug/kg) of GM-CSF (Leucomax) and the remaining seven (Group II) received only an additional dose of HB vaccine. Anti-HBs was determined by Abbott's ELISA kit, and titre above 10 m IU/mL was considered as protective. In Group I, sero-protective titres were obtained in 11 out of 12 (91.6%) patients, whereas in Group II none of the patients achieved sero-protection (p < 0.001). The sero-conversion rate improved from initial 62% (31/50) to overall 84% (42/50) after the use of GM-CSF. There were no adverse events noted with the use of GM-CSF. At one year, 24 out of 32 (75%) who were sero-protected earlier continued to remain sero-protected. This study indicates that GM-CSF is a potent HB vaccine adjuvant for sero-conversion in primary non-responders.

• Dialysis
• GM-CSF
• Hepatitis B vaccine

INTRODUCTION

Hepatitis B (HB) virus infection is moderately endemic in India. The HBV carrier frequency varies from 2% to 7% with an average of 4% [[1]]. The frequency of HBsAg positivity has been reported to be higher among ESRF patients undergoing hemodialysis with the prevalence rate varying in India from 7.6% to 30% [2-3]. Moreover, the mortality due to liver disease among HBsAg positive ESRF patients on hemodialysis is reportedly higher than in HBsAg negative patients and this is especially so after renal transplantation [[2]], [[4]]. Hence, there is a need to prevent HBV infection by effective vaccination among ESRF patients.

The transmission of HBV infection has reduced in the last decade or so because of regular screening for HBsAg, vaccination of high risk groups and treating the asymptomatic HBV carriers with home HD, CAPD or isolated dialysis. Still, HBV infection in HD patients remains an important cause of concern in a developing country like India. Immunization against HBV infection in ESRF stimulates an antibody response only in 30–50% of patients compared to 96–99% in normal population [[2]], [5-8].Not only is the immunological response to HB vaccine low in chronic renal failure population, but the duration of effective protection by antibodies is also short [[9]]. Hence, to improve the efficacy of HB vaccine in ESRF patients, doubling the dose to 40 ug along with increasing number of dosages to four at frequent intervals (0, 1, 2, 6 or 0, 1, 2, 12 monthly schedule) has been advocated [[10]].

To further improve the efficacy of Hepatitis B vaccine various adjuvants that have been tried so far with variable success include erythropoietin, zinc, interleukines, interferon alfa and GM-CSF [11-14].We studied the efficacy of adding GM-CSF to HBV vaccine who were primary non-responders to the usual HB vaccination protocol in ESRF patients.

SUBJECTS AND METHODS

A total of 50 consecutive adult patients who entered our hemodialysis (HD) program during the period of January 1996 to July 1998 were prospectively included. Those included were ESRF patients on regular HD, not vaccinated earlier against HB, and who were both HBsAg and anti-HBs negative. These patients were given 3 doses of recombinant HBV vaccine at 0, 1, 2 month schedule at 40 ug per dose. ESRF was related to CGN1^*[[14]], diabetes (23), ADPKD [[2]], CIN [[8]] and others [[3]]. Patients with active hepatitis, pregnancy, malignancy, HIV infection and any other immuno-compromised state were excluded.

All patients received an average of two HD per week. All patients received 2000–4000 units of erythropoeitin subcutaneously, per week. Each patient had once a month evaluation of the parameters of dialysis adequacy, liver function, iron stores and HBsAg status. Dialyzers were reused as a routine in our dialysis unit.

HBsAg and anti-HBs antibodies were determined using an enzyme linked immunoassay (ELISA) kit. Anti-HBs was analyzed using commercially available AUSAB EIA KITS of Abbott Laboratories. The antibody titres of more than 10 mIU/mL was considered sero-protective as per international standard. The geometric mean titre was calculated in positive sera only.

Anti-HBs titres were checked after four weeks of the third dose. Primary non-responders (defined as patients who failed to develop protective antibody titres after three doses) were given an additional dose of recombinant HB vaccine with or without GM-CSF (Groups I & II, respectively) in a non-randomized fashion. Group I patients received 300 ug (5–6 ug/kg) of GM-CSF (Leucomax) sub-cutaneously in the deltoid region along with an additional intramuscular 40 ug of HBV vaccine, intramuscularly at an adjacent site in the deltoid region simultaneously. Group II (non GM-CSF) received only an additional (fourth) dose of 40 ug recombinant HB vaccine. AntiHBs titres were checked after four weeks of this additional dose. Secondary responders were defined as patients who achieved protective anti-HBs levels after subsequent vaccination with or without GM-CSF as adjuvant. Anti-HBs titres was tested at one year.

The results are expressed as mean ± standard deviation. Statistical analysis was done using Chi Square tests and Student t test. p value of less than 0.05 was taken as significant.

RESULTS

Protective antibody titres developed in 31 patients (62%) after 3 doses each of 40 ug of recombinant HB vaccine with geometric mean titres (GMT) of 181.6 mIU/mL (Table 1). There was no difference in various baseline characteristics to predict response with HBV vaccine between primary responders and primary non-responders. Antibody titres persisted in 17 out of 25 (68%) tested at one year in primary responders with a GMT of 64.95 mIU, which was a significant drop from baseline (p < 0.001).

Table 1. Comparison of Patient Characteristics of Primary Responders & Non-responders

	Primary Responders	Primary Non-responders
Numbers of patients	31	19
Sex (M:F)	20:11	12:07
Mean age (yrs)	48.19 ± 10.7	46.25 ± 15.5
Mean Hb (gm/dL)	8.2 ± 1.4	7.8 ± 1.74
Antibody titres (GMT) mIU/L	181.6	–
Basic disease (diabetic)	12	7

Nineteen (38%) out of 50 patients were primary non-responders to 3 doses of HBV vaccine. Twelve (63%) of the primary non-responders received GM-CSF with an additional fourth dose of HBV vaccine (Group I). The remaining 7 (37%) patients who did not receive GM-CSF (Group II) were administered only an additional (fourth) dose of 40 ug of HBV vaccine.The baseline characteristics of the two groups were comparable (Table 2). The response rate in Group I (secondary responders) was highly significant (p < 0.001) compared to group II (Table 2). There was no additional benefit observed with an extra dose of HBV vaccine alone. HB vaccine and the adjuvant GM-CSF (Leucomax) were well tolerated without any major local or systemic adverse effects. Follow up titres in 7 of 11 secondary responders available showed persistence of seroprotection in all despite a drop of GMT to 50.5 mIU (p > 0.l).

Table 2. Patient Characteristics in Primary Non-responders Receiving Additional Treatment

	Group I (HB Vaccine + GM-CSF)	Group II (HB Vaccine)
Numbers of patients	12	7
Mean age (yrs)	47.8 ± 17.1	43.4 ± 13.1
Males	10	3
Basic disease (diabetes)	3	4
Responders	11	0
Antibody titres (GMT) mIU/L	65.1	–

The success of HB vaccination improved from an initial 62% (31/50) to overall 84% (42/50) with the use of GM-CSF. Antibody titres in secondary responders with GM-CSF was significantly lower (p < 0.001) compared to primary responders (Tables 1 & 2). At one-year follow up, 8 out of 32 (25%) patients lost their protective antibody titres (Table 3). There was a drop of antibody titres from the baseline in both primary and secondary responders. All patients who received GM-CSF showed insignificant drop of antibody titres at one year (p > 0.1).

Table 3. Antibody Titres at 1 Year

	N	Persistent Protection	GMT (Mean)
Primary responder	25	17 (68%)	64.95
Secondary responders	07	07 (100%)	50.55

DISCUSSION

It is well established that patients receiving HD have an increased risk of acquiring HBV infection. Such infections frequently become chronic resulting in significant morbidity and mortality from advanced liver disease while the patients are on dialysis and also following renal transplantation [[2]], [[4]].There is also an increased risk of transmission of HBV to close contacts. Therefore, such dialysis dependent patients must be considered for an effective vaccination strategy [[10]].

With the current vaccination schedule of 40 ug of HB vaccine at 0, 1, 2 and 6 months, only about 50% of vaccinated patients develop protective antibody titres [5-6]. The present study showed that 62% are primary responders. With the use of GM-CSF in primary non-responders the overall success improved to 84%. The duration of protection is often short lasting and nearly 40% lose their sero-protective titres in long term follow up over 3 years [[9]]. In our study, 75% (24/32) had detectable sero-protection at 1 year.

The poor sero-conversion after recombinant HB vaccination in ESRF patients has been attributed to various defects in the immune system. In-vitro studies have established that ESRF patients undergoing regular HD exhibit impairment of immune potential that involve immune system at various levels; namely the antigen presenting cells (APC), various T-cell subsets, T-cell/B-cell co-operation, B-cell function, cytokine production and complement cascade [15-16]. Such defects can be partially overcome with clinical benefits by administration of IL-2 or GM-CSF [[12]], [[18]].

GM-CSF acts by modifying the function of the macrophage. The reported mechanism of actions of GM-CSF are: activating the generation of dendritic cells, activating APCs and increasing the expression of Class II MHC molecule in APC. These enhance antigen presenting activity and thereby augment the primary antibody response through total functional enhancement of APC [[14]].

This study highlights the efficacy of GM-CSF along with recom- binant HB vaccine in dialysis dependent patients not responding to usual vaccination schedule (Table 2). An earlier study on healthy human volunteers has shown quicker development of protective antibody titres with GM-CSF used as an adjuvant along with a single dose of HB vaccine [[17]] and was found useful even in healthy primary non-responders [[18]]. Others have reported similar experience on small numbers of ESRF patients using GM-CSF with beneficial effects [[13]], [19-20]. Hess and colleagues, in their pilot study of 15 patients on HD, who were non-responsive to three doses of 40 ug each of HB vaccine, used three different dosage of GM-CSF (0.5, 5 and 10 ug/kg). The seroconversion rate reported was 7 of 15 (50%) with the best response (4 out of 5) at 5 ug/kg of GM-CSF. Severe hypotension was observed in their study only at 10 ug/kg of GM-CSF [[13]]. The present study confirms that GM-CSF in the dose of 5–6 ug/kg (300 ug) enhances the antibody response in primary non-responders. Moreover, with this dose no major adverse effects were observed.

We used GM-CSF as an adjuvant only in primary non-responders. Others have used GM-CSF in small number of patients with the first dose of HB vaccine with beneficial results [19-20]. Though our results show that 11 of 12 primary non-responders developed protective antibody titres after adjuvant therapy, the titres were significantly lower than primary responders (p < 0.001). Such low titre response is anticipated in those who are unable to mount an effective antibody response [[13]]. At one year follow up, 7 secondary responders (Group I) tested had intact sero-protection (Table 3).

A major drawback of this study is the small number of patients and non-randomized nature of study design. Despite its limitation, development and persistence of sero-protection in primary non-responders indicates the utility of such vaccination strategy.

Although this study confirms the utility of GM-CSF in HB sero-conversion in dialysis dependent patients a number of issues warrant further investigation. Larger and long-term controlled studies are required to confirm the efficacy of GM-CSF in vaccine response.

ACKNOWLEDGMENT

We gratefully acknowledge the contribution of Mr. P.V.L.N. Raju, Clinical Research Executive of Shanta Biotech for statistical assistance.

Notes

^*CGN = chronic glomerulonephritis, ADPKD = adult dominant polycystic kidney-disease, CIN = chronicintrestialnephritis.