Humoral immune response and delayed type hypersensitivity to influenza vaccine in patients with diabetes mellitus

Diabetologia, Jun 1987

The antibody response and delayed type hypersensitivity reaction to commercially available trivalent influenza vaccine in 159 patients with diabetes mellitus was compared with response and reaction in 28 healthy volunteers. A correction for prevaccination titres was made. No differences were found between diabetic patients and control subjects with respect to antibody response to the three vaccine strains as measured by the difference between geometric mean titres of post- and prevaccination sera. In Type 1 (insulin-dependent) diabetic patients the incidence of non-responders to two vaccine components was significantly increased (p< 0.05). The delayed type hypersensitivity reaction to influenza antigen was significantly decreased in patients with high concentrations of glycosylated haemoglobin (p<0.01). These findings suggest a role for impaired immune response in the increased influenza morbidity and mortality in patients with diabetes mellitus. Implications for therapy and vaccination strategy are discussed.

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Humoral immune response and delayed type hypersensitivity to influenza vaccine in patients with diabetes mellitus

Diabetologia Humoral immune response and delayed type hypersensitivity to influenza vaccine in patients with diabetes mellitus R. J. A. D i e p e r s l o o t 0 K. P. Bouter 0 W. E. P. Beyer 0 J. B. L. H o e k s t r a 0 a n d N. Masurel 0 0 1Department of Virology and WHO Influenza Centre, University Hospital Dijkzigt , Rotterdam , and 2Department of Internal Medicine, Diakonessen Hospital , Utrecht , The Netherlands Summary. The antibody response and delayed type hypersensitivity reaction to commercially available trivalent influenza vaccine in 159 patients with diabetes mellitus was compared with response and reaction in 28 healthy volunteers. A correction for prevaccination titres was made. No differences were found between diabetic patients and control subjects with respect to antibody response to the three vaccine strains as measured by the difference between geometric mean titres of post- and prevaccination sera. In Type 1 (insulin-dependent) diabetic patients the incidence of non-responders to two vaccine components was significantly increased (p< Diabetes mellitus; influenza; delayed type hypersensitivity; vaccination; immunity 9 Springer-Vedag 1987 Infections with influenza carry a high m o r b i d i t y a n d mortality rate in patients with diabetes mellitus [ 1-3 ]. T h e increased risk o f c o m p l i c a t i o n s in these patients is generally ascribed to the o c c u r r e n c e o f diabetic ketoacidosis [ 4 ] a n d s e c o n d a r y bacterial infection, m a i n l y b y S t a p h y l o c o c c u s aureus [ 5 ]. Patients with diabetes mellitus are often carriers o f S t a p h y l o c o c c u s aureus, a n d t h e y h a v e b e e n s h o w n to h a v e an i m p a i r e d i m m u n e res p o n s e to this m i c r o - o r g a n i s m [ 6, 7 ]. I n o r d e r to p r e v e n t these complications, a n n u a l v a c c i n a t i o n o f diabetic patients is r e c o m m e n d e d . To a c c o m p l i s h p r o t e c t i o n against influenza, vaccination s h o u l d i n d u c e high a n t i b o d y titres against the viral h a e m a g g l u t i n i n [ 8 ]. S i m u l t a n e o u s l y stimulated cellular immunity, t h o u g h not protective, m i g h t contribute to the r e c o v e r y f r o m infections with influenza viruses [ 9 ]. P o o r a n t i b o d y r e s p o n s e to influenza vaccination has b e e n d e m o n s t r a t e d in various risk groups, such as renal t r a n s p l a n t patients [ 10 ], patients with m a l i g n a n t diseases [ 11, 12 ] a n d in the a g e d [13]. I n o r d e r to evaluate the i m m u n e r e s p o n s e to influe n z a antigen in b o t h Type 1 (insulin-dependent) a n d Type 2 ( n o n - i n s u l i n - d e p e n d e n t ) diabetic patients, we studied the a n t i b o d y p r o d u c t i o n a n d d e l a y e d type hypersensitivity reaction after v a c c i n a t i o n with a trivalent influenza vaccine. 0.05). The delayed type hypersensitivity reaction to influenza antigen was significantly decreased in patients with high concentrations of glycosylated haemoglobin (p<0.01). These findings suggest a role for impaired immune response in the increased influenza morbidity and mortality in patients with diabetes mellitus. Implications for therapy and vaccination strategy are discussed. Subjects and methods Subjects Patients studied were attending the outpatient clinic of the Department of Internal Medicine of the Diakonessen Hospital, Utrecht, The Netherlands. Patients were considered to be Type I if there had been documented ketoacidosis and/or abrupt onset of symptoms requiting insulin therapy at age < 40 years and Type 2 if there had been protracted treatment with diet or oral therapy at age > 40 years. The study population consisted of 27 patients with Type 1 diabetes mellitus, 18 men and 9 women (mean age 39.3+ 13.6 years, mean duration of disease 16.5_+14.0 years) and 120 patients with Type 2 diabetes mellitus, 51 men and 69 women (mean age 65.3_+10.0 years, mean duration of disease 10.3+7.1 years). Among 12 patients, 5 men and 7 women (mean age 61.9_+7.4 years), the type of' diabetes was unknown. In Type 1 diabetic patients, 5 had known cardiovascular complications, 3 were treated for retinopathy and 1 had marked neuropathy. Among Type 2 patients 37% were more than 10% overweight and 25% had major cardiovascular complications. Retinopathy was diagnosed in 16 and neuropathy in 13% of Type 2 diabetic patients. Control subjects were 28 healthy volunteers, 13 men and 15 women (mean age 50.8_+17.0 years). Participants were excluded if they were allergic to egg protein or when febrile on the day of vaccination. Written consent was obtained from all participants and approval for the study was obtained from the Ethical Committee of the University Hospital Dijkzigt. Vaccine: dosage and administration Trivalent purified whole virus influenza vaccine (Duphar-Nederland, Amsterdam, The Netherlands) containing 10 Ixg haemagglutinin (HA) A/Philippines/2/82 (H3N2), 10 ~g HA A/Chile/I/83 (HiNt) and 15 gg HA B/USSR/100/83 was administered in 0.5 ml doses intramuscularly in the upper arm. To induce a delayed type hypersensitivity reaction, an 0.t ml dose of the same vaccine (diluted 1 :1 with phosphate buffered saline) was inoculated into the skin of the volar aspect of the forearm. Laboratory investigations and calculations Blood samples were obtained prior to administration of vaccine and again 14 days later. Sera were separated immediately after blood collection and clotting and stored at - 2 0 ~ until titration. Influenza strains were propagated in embryonated hen's eggs. Because of the low avidity of the influenza B virus, infectious egg fluids of this strain were treated with aether according to Berlin et al. [ 14 ] and the watery phase was used in the serologic tests. Serum haemagglutination inhibition (HI) titres were determined twice by standard methods [ 15 ]simultaneously in pre- and post-vaccination sera. Titres were expressed as reciprocals of the dilution showing 50% haemagglutination inhibition with 3 haemagglutination units of the antigen. From the results of the two determinations per serum and per antigen, the geometric means were used for further calculations. Negative titres (< 9) were arbitrarily regarded as 5. With the method used, protection against influenza is thought to be associated with an HI titre of 100 for influenza A [ 8 ]. No protection threshold is known for aether-treated influenza B strains. For this study an HI titre of 100 was assumed to be protective. Among diabetic patients and control subjects, those with prevaccination titres above 100 were excluded separately for each antigen. The serologic response upon vaccination was expressed using the following criteria: the response rate (i. e. the proportion of subjects with a 4-fold or greater titre increase after vaccination); the protection rate (i. e. the proportion of subjects exceeding the threshold titre of 100 after vaccination); the mean-fold increase (i. e. the difference between the logarithmated geometric mean titres of post- and prevaccination sera). Glycosylated haemoglobin The percentage of glycosylated haemoglobin (HbAac) on the day of vaccination was determined by a commercially available column test (Bio Rad Laboratories, Richmond, Calif, USA). In short, a small quantity of whole blood is mixed with a haemolysis reagent. An aliquot of the haemolysate is then applied to a weakly acidic cation exchange resin in a disposable column. The HbAla and HbAlb fractions are first eluted by adding a buffer. The HbAlc fraction is then eluted separately by adding a second dilution/developing reagent. The relative % concentration of HbAlc is determined spectrophotometrically. Delayed type hypersensitivity reaction (D THR) DTHR was read after 24 h. Quantification of the test was achieved by calculating the area of induration as the product of two diameters at right angles. Diameters were measured as described previously by Sokal [ 17 ]. Statistical analysis Data are presented as mean+ SD. Differences in qualitative measures were tested for significance by the chi-square test, and in quantitative measures by the Wilcoxon rank test. Results Seroresponse T h e o u t c o m e o f the serologic d e t e r m i n a t i o n s w a s calc u l a t e d f o r t y p e o f diabetes mellitus a n d f o r the therap e u t i c regimen. Results are p r e s e n t e d f o r the three vaccine strains s e p a r a t e l y in Tables 1 - 3 . A l t h o u g h patients with Type 1 diabetes a n d t h o s e with T y p e 2 diabetes t r e a t e d with a diet o n l y t e n d e d to h a v e l o w e r a n t i b o d y r e s p o n s e s after v a c c i n a t i o n as c o m p a r e d to c o n t r o l subjects, differences in m e a n - f o l d increase were n o t statistically significant. T h e established p r o t e c t i o n rate w a s h i g h f o r the H 3 N 2 strain (Table 1), r e a c h i n g 90% in c o n t r o l subjects a n d 85% in d i a b e t i c patients. Prot e c t i o n rates f o r the o t h e r t w o v a c c i n e c o m p o n e n t s , however, were c o n s i d e r a b l y l o w e r : 66 a n d 64% f o r H 1 N a a n d 50 a n d 57% f o r the i n f l u e n z a B strain (control subjects a n d patients, respectively). D i f f e r e n c e s w e r e n o t statistically significant. I n c o m p a r i s o n with c o n t r o l subjects, the i n c i d e n c e o f patients s h o w i n g a 4-fold o r greater titre rise was substantially l o w e r in T y p e 1 diabetes f o r the H 3 N 2 a n d i n f l u e n z a B v a c c i n e c o m p o n e n t s (100 vs 78% a n d 80 vs 44%, respectively, p < 0.05). A significantly l o w e r i n c i d e n c e o f patients with a 4-fold o r greater titre increase to the i n f l u e n z a B strain was also s h o w n f o r patients t r e a t e d with insulin, a m a j o r p a r t o f w h o m h a d T y p e I diabetes (46 vs 80% in c o n t r o l subjects, p < 0.01). F o r patients treated with diet only, the i n c i d e n c e o f patients with a 4-fold o r greater titre increase w a s sign i f i c a n t l y l o w e r for the H 3 N 2 c o m p o n e n t (78 vs 100% in c o n t r o l subjects, p < 0 . 0 5 ) . T h e r e was n o c o r r e l a t i o n b e t w e e n a n t i b o d y p r o d u c t i o n o r r e s p o n s e rate a n d the c o n c e n t r a t i o n o f HbAtc. Delayed type hypersensitivity reaction (DTHR) I n o r d e r to establish a c o r r e l a t i o n b e t w e e n the D T H R a n d the m e t a b o l i c state, all 159 patients were divided into t w o g r o u p s a c c o r d i n g to the c o n c e n t r a t i o n o f glyc o s y l a t e d h a e m o g l o b i n : H b A t c 4 - 6 . 5 % (within n o r m a l limits), a n d > 6.5%. T h e largest i n d u r a t i o n was d e m o n s t r a t e d in c o n t r o l subjects: 360 m m 2 (_+ 246). I n patients with H b A t values within n o r m a l limits ( H b A I < 6 . 5 % ) , the D T H R w a s similar to that in c o n t r o l subjects. I n c o m p a r i s o n with c o n t r o l subjects, the D T H R in patients with a n H b A a o > 6 . 5 % was significantly d e c r e a s e d ( p < 0 . 0 1 ) . Results are s h o w n in F i g u r e 1. Discussion F r o m a p r e v i o u s s t u d y it w a s c o n c l u d e d t h a t patients with well c o n t r o l l e d diabetes mellitus r e s p o n d n o r m a l ly to i n f l u e n z a i m m u n i s a t i o n . T h e p o p u l a t i o n studied, h o w e v e r , was small a n d p r e v a c c i n a t i o n titres were cons i d e r a b l y h i g h e r in c o n t r o l subjects, f o r w h i c h n o corr e c t i o n was m a d e [ 18 ]. I n the p r e s e n t s t u d y a c o r r e c t i o n w a s i n c l u d e d f o r p r e v a c c i n a t i o n titres. It is s h o w n that, at least in patients with Type i diabetes, there is a n inc r e a s e d i n c i d e n c e o f n o n - r e s p o n d e r s to t w o o f the t h r e e v a c c i n e c o m p o n e n t s . H u m o r a l i m m u n e r e s p o n s e Data of 12 patients whose type of diabetes was unknown and of 2 patients with both insulin and oral therapy are not shown Data of 12 patients whose type of diabetes was unknown and of 2 patients with both insulin and oral therapy are not shown, a Different from control subjects p < 0.05; bp < 0.01 9 18 94 78a 4 23 56 44a Type 1 (n = 27) 7.5(+1.6) 4 23 73 70 36 84 83 87 62 67 25 95 58 65 17 40 85 88 6 51 70 69 8 49 63 76 2 18 77 78a 1 19 47 58 6 14 35 65 29 31 88 87 7 73 64 58 19 61 63 46b Type I (n = 27) Type 2 (n = 120) Oral therapy (n = 57) Diet (n = 20) Insulin (n = 80) 7.4(+1.5) 7.9(+1.4) 8.0(+1.5) 7.0(+l.1) 8.0(_+1.5) Control subjects (n= 28) 8 20 90 100 Control subjects (n = 28) 4 24 66 67 4 24 50 80 Control subjects (n = 28) 5.0(+0.5) Number of subjects with prevaccination titre > 100 Mean HbA~c% (+_SD) 5.0(+0.5) Subjects studied Mean-fold increase (___SD) % of subjects with postvaccination titre > 100 % of subjects with 4-fold or greater titre increase Number of subjects with prevaccination titre > 100 Mean HbA1o% ( + S D ) Subjects studied Mean-fold increase ( + S D ) % subjects with post-vaccination titre > 100 % subjects with 4-fold or greater titre increase 85 86 64 65 Diabetic patients Total (n = 159) 14 7.7(+1.5) 145 Diabetic patients Data of 12 patients whose type of diabetes was unknown and of 2 patients with both insulin and oral therapy are not shown, a Different from controls p < 0.05 to influenza vaccination has been shown to be impaired in the elderly [ 13 ]; however, as control subjects (mean age 50.8_+ 17.0 years) were older than Type 1 diabetic patients (mean age 34.4_+ 13.6 years), age cannot be held responsible for the increased incidence of non-responders among Type 1 patients. Antibody formation against the influenza antigen is a T-cell dependent phenomenon. In experimental animals the humoral immune response is impaired if the helper effect o f T cells is lacking [ 19 ]. In patients with Type I diabetes, T-cell depletion has recently been demonstrated [ 20 ]. This may explain the increased incidence of non-responders to influenza antigen, while antibody response to pneumococcal polysaccharide, which may proceed independently of T-cell help, is not decreased [ 21 ]. The number of patients unable to acquire a protective antibody level against the influenza B and H1N1 vaccine components is substantial. This is an important outcome, considering the high incidence o f other risk factors such as cardiovascular diseases, especially in elderly diabetic patients. Barker and Mullooly [ 1 ] showed that influenza mortality is highest in patients who have cardiovascular disease in combination with either diabetes or chronic pulmonary disease. Therefore, a booster immunisation after at least 4 weeks seems to be advisable in patients with diabetes mellitus. However, results of booster vaccination in other risk groups are disappointing [ 10, 22 ]. Decreased D T H R to candida in diabetic patients has been previously demonstrated [ 23 ]. In the same study no decreased D T H R was found for a viral antigen (mumps). M a h m o u d et al. [ 24 ] showed that decreased cellular hypersensitivity in diabetic mice could be restored with insulin treatment. Our findings of a decreased D T H R in patients with high HbAlo values and not in patients with HbAtcvalues within normal limits suggest that optimal regulation might restore the D T H R in humans. The function of T cells which mediate the D T H R in influenza infections is not clear. In mice these cells were found in the lungs after infection with an influenza A virus, the concentration of cells being correlated with the amount of virus administered [ 25 ]. For recovery from the infection, however, the cytotoxic T cell and natural killer cell are probably more important [ 9 ]. Until now it was assumed that the main risks of influenza infection in patients with diabetes mellitus lie in the occurrence o f ketoacidosis [ 4 ] and secondary bacterial infection [ 5 ]. From this study it can be concluded that impaired immune response to the influenza virus itself may contribute to increased morbidity and mortality. Acknowledgements. This study was funded by a grant from DupharNederland (Amsterdam, The Netherlands). We wish to thank Mr. R. van Beek and Mr. H. van Driel for technical assistance, Prof. Dr. W. E. Erkelens for comments and Mrs. R. S. Engels-Bakker for preparation of the manuscript. 1. Barker WH , Mullooly JP ( 1982 ) Pneumonia and influenza deaths during epidemics . Arch Intern Med 142 : 85 - 89 2. Housworth J , Langmuir A ( 1974 ) Excess mortality from epidemic influenza 1957-1966 . Am J Epidemiol 100 : 40 - 47 3. 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Thymus dependence of antibody formation and thymus independence of immunological memory . J Exp Med 140 : 1559 - 1570 20. Quinion-Debrie MC , Debray-Sachs M , Dardenne M , Chernichow P , Assan R , Bach JF ( 1985 ) Anti-islet cellular and humoral immunity, T-cell subsets, and thymic function in type 1 diabetes . Diabetes 34 : 373 - 379 21. Lederman MM , Schifman G , Rodman HM ( 1981 ) Pneumococcal immunizationin adult diabetics . Diabetes 30 : 119 - 121 22. Versluis DJ , Beyer WEP , Masurel N , Weimar W ( 1985 ) Influenza vaccination in dialysis and transplant patients . Antiviral Res Suppl . 1 : 289 - 292 23. Plouffe JE , Silva J , Fekety R , Allen JL ( 1978 ) Cell mediated immunity in diabetes mellitus . Infect Immun 21 : 425 - 429 24. Mahmoud AAF , Rodman HM , Mandel MA , Warren KS ( 1976 ) Induced and spontaneous diabetes mellitus and suppression of cell-mediated immunologicresponses . J Clin Invest 57 : 362 - 367 25. Leung KN , Ada GL ( 1980 ) Cells mediating delayed type hypersensitivity in the lungs of mice infected with an influenzaA virus . Scand J Immunol 12 : 393 -400 Received: 10 November 1986 and in revised form: 6 April 1987 Dr. R.J .A. Diepersloot Department of Virology Erasmus University Rotterdam P. O. Box 1738 NL-3000 DR Rotterdam The Netherlands


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Dr. R. J. A. Diepersloot, K. P. Bouter, W. E. P. Beyer, J. B. L. Hoekstra, N. Masurel. Humoral immune response and delayed type hypersensitivity to influenza vaccine in patients with diabetes mellitus, Diabetologia, 1987, 397-401, DOI: 10.1007/BF00292541