A randomized, placebo-controlled study of oral cimetidine as an immunopotentiator of parenteral immunization with a group B meningococcal vaccine

Vaccine,Vol.

15, No. IO, pp. 1144-1146, 1997 Published by Elsevier Science Ltd. Printed in Great Britain 0264-41OW97 $17+0.00

Elsevier PII: 60264-410X(96)00311-8

ELSEVIER

A randomized, placebo-controlled study of oral cimetidine as an immunopotentiator of parenteral immunization with a group B meningococcal vaccine Joseph J. Drabick*T, Douglas B. Tang?, E.E. Moran*, Janet S. Foster5 and Wendell D. Zollinger*

Andrew

F. Trofa$,

Cimetidine (CIM) is an H2-receptor antagonist with a long history of clinical use in peptic ulcer disease. In addition to its inhibitory efSect upon gastric acid secretion, CIA4 can also block histamine-mediated immunosuppression by inhibiting H2 receptors on suppressor T cells. CIA4 results in immunoaugmentation of both cellular and humoral immunity by this mechanism and has been used clinically in the treatment of chronic infectious and neoplastic diseases. We postulated that orally administered CIM, like an adjuvant, could augment the immunologic response to a parenteral vaccine. To test this hypothesis, a randomized placebo (PLB) -controlled, double-blinded study in 14 healthy volunteers was performed using a Group B meningococcal outer membrane protein (OMP) vaccine administered twice, 6 weeks apart. Volunteers were randomized within pairs defined by their screening OMP antibody titers to receive either CIM or PLB which was administered for 5 days, beginning 2 da_ys before each of the two immunizations. All 14 volunteers completed the stat& with excellent compliance. Sera were tested jar anti-OMP and bactericidal antibodies. The groups were comparable in terms of gender distribution, age and baseline anti-OMP titers. Reactogenicity to the vaccine was mild and comparable between groups. There was little efSect of CIM (over PLB) on anti-OMP or functional bactericidal antibody levels over time. Geometric means oj’maximum OMP antibody increase over baseline was 3.3-fold (95% CI: 1.86.3) for CIA4 and 2.4 for PLB (CI: 1.6-3.7). CIA4 had a corresponding 3.9-fold increase (CI: 1.9-8.3) in bactericidal antibody level compared to 2.2 for PLB (CI: 1.4-3.4). We conclude that oral CIM was not eflective as an immunopotentiator of immunization with this group B meningococcal vaccine. 0 1997 Elsevier Science Ltd. Keywords:

cimetidine;

immunoaugmentation;

meningococcal;

vaccine;

There have been many approaches to boosting the immune response to poorly immunogenic vaccines. One approach is to alter the molecular structure so as to increase immune responsiveness. Another is to combine vaccines with adjuvants when administered parenterally to increase their potency. Adjuvants non-specifically *Department of Bacterial Diseases, Walter Reed Army Institute of Research, Washington, DC 20307-5100, USA. TDivision of Biometrics, Walter Reed Army Institute of Research, Washington, DC 203075100, USA. fClinical Trials Section, Walter Reed Army Institute of Research, Washington, DC 20307-5100, USA. §lnfectious Disease Pharmacy, Walter Reed Army Medical Center, Washington, DC 203075000, USA. To whom correspondence should be addressed. (Received 15 May 1996; revised 21 November 1996; accepted 21 November 1996)

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stimulate the local cells of the immune system responding to the injected antigen’. An alternative approach to the local immunoaugmentation induced by an injected adjuvant, would be to boost the immunoresponsiveness of the whole subject such that an effective immune response to the antigen would be more likely to occur. Cimetidine (CIM) is an H2-receptor antagonist with a long history of safe use in gastric acid-mediated gastroesophageal disorders. It is currently available over-the-counter in the United States. Although little appreciated, CIM also exerts immunologic effects via its interaction with H2-receptors on suppressor T-cells’. The subsequent immunoaugmentation abilities are unique to CIM when compared to other H2antagonists3. Enhancement of both humoral and cellular immune response has been demonstrated; these effects

Effect of cimetidine on immunization: have been reviewed in detai14s5. CIM has been used clinically because of these immune effects in the treatment of cancer6 and some infectious diseases such as chronic Herpes virus infection7 and HIV8,9. Recently, it was noted that CIM delivered intraperitoneally as a dose in mice 2 days before imsingle 40 mg kg-’ munization resulted in roughly tenfold higher antibody titers to weakly immunogenic bacterial antigens compared to untreated controls”. To date, this effect has not been demonstrated in humans in a clinically practical fashion. To test the immunoaugmenting potential of CIM, we conducted a small placebo (PLB)-controlled, doubleblinded study to determine if it could induce an enhanced humoral response against an outer membrane protein (OMP)-based Group B meningococcal vaccine in humans. This vaccine had been used in a phase I clinical trial in adult volunteers where the immunogenicity was less than optimal”. Related group B meningococcal OMP-based vaccines have been shown to induce stronger overall antibody responses in young that children than adults1’~13. We have postulated immunologic tolerance may be the reason for the lower immunogenicity of OMP vaccines in adulthood. The tolerization to meningococcal OMP proteins may be induced by asymptomatic colonization with group B meningococci or with commensal Neisseriaciae, the likelihood of which increases with chronologic age. Of interest, CIM had been shown to overcome the immunologic tolerance which had developed to topical dinitrochlorobenzene sensitivity used clinically to treat alopecia areatai4. After 2 days of treatment with oral CIM, the acquired tolerance to the compound was inhibited and full reactivity to the compound was restored. Because of its relatively poor immunogenicity in adults and the potential role of tolerance in mediating this, the group B OMP-based meningococcal vaccine was chosen for study.

MATERIALS

AND METHODS

Protocol development The protocol was developed by the investigators and took place in the Clinical Trials section of the Walter Reed Army Institute of Research (WRAIR), a licensed facility which follows Good Clinical Practices. The study was reviewed and approved by local institutional review boards and the United States Food and Drug Administration. Volunteers Healthy male and female volunteers (military or civilian) ages 18-35, were sought out non-coercively from the WRAIR. Exclusion criteria included:

(1) fever 38°C within a week prior to immunization; (2) history of allergy to any vaccine or HZreceptor antagonist; (3) HIV seropositivity; disease; (4) history of significant organ/system abnormalities on (5) presence of clinically significant screening laboratory tests (complete blood count, routine chemistries); famotidine or other (6) any use of CIM, ranitidine, H2-receptor antagonist within a year of entry;

J.J. Drabick et al.

(7) pregnancy (positive [beta-human chorionic gonadotropin] p-HCG within 2 days prior to receipt of study drug); (8) prior receipt of any Group B meningococcal vaccine; (9) history of meningococcal disease; IO) use of any prescription drug at the current time.

Vaccine and drugs/immunization

schedule

The meningococcal OMP vesicle vaccine, lot 5274P2, was produced by direct extraction of whole cells of strain 8765 (B:15:P1.3:L3,7,8) rec( -) with 0.5% deoxycholate. The outer membrane vesicles were isolated by differential centrifugation including ultracentrifugation twice through a sucrose shelf. The vaccine was adsorbed to aluminum hydroxide (0.76 mg aluminum per 0.5 ml dose) and stabilized with 5% sucrose in normal saline. Thimerosal at 0.01% was added as a preservative. Residual LOS in the vaccine was about 5% relative to protein’ ‘. CIM elixir 300 mg in 5 ml (Tagamet brand, SmithKlein Beecham, Philadelphia, PA) was aliquotted into single dose opaque containers and sealed. The PLB was the vehicle used in the Tagamet elixer without active drug and was sight and taste matched to the active drug. The PLB was similarly packaged. All volunteers were immunized in the deltoid muscle of the non-dominant arm with 20 ug of vaccine in 0.4 ml saline with alum at 0 (primary) and 6 weeks (booster). Beginning 2 days prior to each scheduled immunization, CIM or PLB was taken four times a day for 5 days. The CIM dose chosen (300 mg qid) has been shown to have immunoaugmenting efficacy in humans4,14.

Study design The trial was designed as a randomized, PLBcontrolled, double-blind study to test for large effects of CIM on humoral immune function as measured by quantitative and functional antibody production. Only large effects would be considered worthwhile from a practical point of view. Fourteen healthy male and female volunteers who met all inclusion/exclusion criteria were entered into the study after informed consent was obtained. Volunteers were pair matched based on their screening baseline OMP titers (two highest,..., two lowest); and then, using a computer generated list, were randomized to receive either CIM (n=7) or PLB (n=7). Assignments were sealed in opaque envelopes and the code was maintained by the study statistician and medical monitor. Matching was used to control for subject differences in baseline OMP titers since it is known that some individuals possess significant anti-meningococcal antibodies’5,‘6. Total sample size (n=14) was selected to detect a difference (PLB vs CIM) in mean (max) titer of 1.5 standard deviations (S.D.; 80% power, one-sided a=5%) and did not account for possible increase in power due to reduced variability due to matching17.

Measurements Reactogenicity to immunization was monitored by interview, questionnaire, physical examination, and a temperature recording during the first 48 h following

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each immunization. Volunteers were instructed to report any symptom that they may experience and were provided with disposable thermometers. Compliance (drug and PLB) was ascertained using container counts. Sera for determining immunologic response were drawn at prevaccination (day 0) and at target times of 3, 6, 9, 12, and 21 weeks after the first immunization. Protocol allowance was *3 days. All sera (all time periods) were stored and tested in simultaneous assays (to minimize inter-assay variation) for antibodies against OMP and outer membrane complex (OMC) by ELISA (IgG, IgA, IgM) and for serum bactericidal activity (CIDAL) against the 8765 (B:P15:P1.3,L3,7,9) strain of Neisseriu meningitidis as described previously in detail’8-‘9. The OMC is a preparation of native outer membrane vesicles from group B meningococci from which OMP is prepared and contains iipopolysaccharide in addition to the OMP. The serum bactericidal assay measures complement-mediated lysis by antimeningococcal antibodies and correlates with protection against invasive meningococcal disease”. All measurements were performed blind to group assignment.

Statistical analysis Boxplots were used to graphically compare (CIM vs PLB) the magnitude and time course of antibody response to the OMP and OMC and for serum bactericidal activity. Repeat measures analysis of variance (ANOVA) was used as the statistical framework for assessing overall group differences (CIM vs PLB), time effects (repeat measure factor), and group by time interactions”. This analysis took into account the matching on screening OMP antibody levels. Because of volunteer no-shows, some sera were not available at week 9 (two CIM, three PLB) Analyses ignoring week 9 and using missing values estimated as the mean of proceeding and following periods were done and yielded similar results. ANOVA results using estimated values are reported. Paired t-tests were used to compare post vaccination maximum (and mean) antibody level between groups or changes over baseline. Corresponding 95%) confidence intervals were computed. In all analyses, data were examined for possible effects of suspected outliers and transformed (log) to reduce skewness. The Minitab statistical software system was used for all calculations”.

RESULTS Two of the original volunteers randomized withdrew before beginning the study and were replaced by alternates. All volunteers beginning treatment successfully completed the study without incident. One volunteer missed the booster immunization and drug associated with it due to a schedule conflict but otherwise completed the study. This volunteer was found to be in the PLB group when the code was broken. CIM and PLB were comparable with respect to gender (CIM: 4F/3M: PLB: 3F/4M), median age (CIM: 23 years; PLB: 29 years), and mean (S.D.) baseline anti-OMP titer [CIM: 1.56 (1.55) ug ml-‘; PLB: 1.59 (1.53) ug ml-‘]. Compliance with the oral medications before both primary and booster immunizations was excellent (95%) in both the CIM (272/280 doses) and PLB (253/260 doses)

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Figure 1 Boxptot showing specific antibody levels by ELISA (total IgG, IgA, IgM) to OMP against the 8765 (B:P15:P1.3,L3,7,9) strain of group B N. meningitidis. Boxplot shows first and third quartiles (bottom, top of box), median (symbol within box), and extremes (minimum, maximum points of extended line). Closed (open) circles are the CtM (PLB) groups

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Figure 2 Boxplot showing specific antrbody levels by ELISA (total IgG, IgA, IgM) to OMC against the 8765 (B:PlS:P1,3,L3,7,9) strain of group B N. meningifidis. Boxplot shows first and third quartiles (bottom, top of box), median (symbol within box), and extremes (minimum, maximum points of extended line). Closed (open) circles are the CIM (PLB) groups

groups. Reactogenicity was minimal in both groups with transient mild tenderness at the injection site noted equivalently in the two groups (CIM 717; PLB 6/7). The booster immunization was not associated with more reactogenicity in either group. The comparative results of the ELISA tests for antibodies against OMP, OMC and the CIDAL between CIM and PLB are summarized in Figures I-3, respectively. For all three antibody determinations, the overall difference in geometric mean levels between CIM and PLB was small and not statistically significant [max F(1,6)=0.93; min PzO.37 for OMC]. The time courses were essentially parallel (no group by time interaction). Both CIM and PLB showed a rise over baseline (time 0) which accounted for a statistically significant time effect for all three types of antibodies [min F(5,6)=5.78; mm P
Effect of cimetidine on immunization:

Likewise, the problem of preexisting immunity and the issue of tolerance as in the case of group B meningococci may be difficult to overcome. The CIM immunoaugmentation may therefore be more effective when given adjunctively with vaccines to which the host is essentially naive, such as the hepatitis B or malaria peptide-based vaccines. Although CIM has been demonstrated to be an effective immunoaugmenting agent, other compounds are available such as levamisole2’ which may be more efficacous in this particular utilization. For these reasons, we feel that this approach should be studied further.

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Figure 3 Boxplot showing the functional serum bactericidal antibodv (CIDAL) aaainst the 8765 (B:P15:P1.3.L3.7.9) strain of aroup B N. r&&ingiti&.Boxplot shows f:lrst and third quartiles (bottom, top of box), median (symbol within box), and extremes (minimum, maximum points of extended line). Closed (open) circles are the CIM (PLB) groups

volunteers receiving PLB were 2.4 for OMP (95”/0 CI: 1.6-3.7) 2.9 for OMC (CI: l&4.6), and 2.2 for CIDAL (CI: 1.4-3.4). Differences between the CIM and PLB groups in maximum increase were not significant. Similar results were obtained for increase in mean level (mean post-immunization level/baseline). The results in terms of the humoral response and reactogenicity are comparable to the previous experience with the vaccine ’ ’ . DISCUSSION The use of CIM did not result in clinically significant augmentation of the humoral response to the Group B meningococcal OMP vaccine at the dosage and frequency used in this study. Although a larger study may have detected smaller effects due to CIM, such small overall effects or good effects in a subset of the population render CTM less practical as an immunopotentiating agent of vaccination for a general population. The dosage of CIM which was employed is known to have significant immunomodulating effects from previous It is unlikely that a higher dose or clinical experience4.“. duration of treatment would have resulted in increased efficacy. Likewise, therapy more than four times a day for 5 days around the time of immunization becomes unwieldy and impractical. This approach of attempting to boost the immunologic response to a vaccine through a concurrently administered systemic immunoaugmenting agent is to our knowledge a novel one. Although this approach failed for CIM and the group B meningococcal vaccine -used in this study, it is important to state as a caveat that this was just one vaccine; CIM may have a measurable effect on immune responsiveness to other vaccines in humans. We were concentrating on the humoral responses to this vaccine since protection in meningococcal infection is mediated by antibody and complement. CIM affects both the humeral and cellular arms of the immune system. Immunoaugmentation by CIM may be more readily demonstrated for vaccines in which a cellular immune response is desired such as with the BCG (Bacillus Calmetle-Gutrin) tuberculosis vaccine.

ACKNOWLEDGEMENTS The views of the authors do not purport to be those of the United States Army or the Department of Defense. This study was approved by the Human Use Committees of the Walter Reed Army Institute of Research and the US Army Surgeon General. We acknowledge the expert assistance of Linda Cortese DPharm and J. Bruce McClain MD.

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Rosenquist, E., Harthug, S., Froholm, L.O., Hoiby, E.A., Bovre, K., Zollinger, W.D. Antibody responses to serogroup B meningococcal outer membrane antigens after vaccination and infection. J. Clin. Microbial. 1988, 26, 1543-l 548 Zollinger, W.D., Boslego, J., Froholm, L.O., Ray, J.S., Moran, E.E. and Brandt, B.L. Human bactericidal antibody response to meningococcal outer membrane protein vaccines. In: Gonococci and Meningococci (Eds Poolman, J.T., Zanen, H.C., Meyer, T.F., et a/.). Kluwer Academic Publishers, Dordrecht, Netherlands, 1988, pp. 209-217 Machin, D. and Campbell, M.J. Statistical Tables for the Design of Clinical Trials. Blackwell Scientific Publications, Boston, 1987 Zollinger, W.D., Brandt, B.L. and Tramont, E.C. Immune response to Neisseria meningitidis. In: Manual of Clinical Immunology (Eds Rose, N.R. and Friedman, H.), 3rd Edn. American Society for Microbiology, Washington, DC, 1986, p. 346

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Moran, E.E., Brandt, B.L., Zollinger, W.D. Expression of the L8 lipopolysaccharide determinant increases the sensitivity of Neisseria meningitidis to serum bactericidal activity. infect Immunol. 1994,62, 5290-5295 Goldschneider, I., Gotschlich, E.C. and Artenstein, M.S. Human immunity to the meningococcus I. The role of humoral antibodies. J. Exp. Med. 1969,129,1307-1326 Glantz, S.A. and Slinker, B.K. Primer ofApplied Regression and Analysis of Variance. McGraw-HitI, New York, 1990 Minitab Inc. Minitab Reference Manual (Version 10 Xtra for Windows). State College, PA, 1994 Drews, J. The experimental and clinical use of immunemodulating drugs in the prophylaxis and treatment of infections. infection 1985, 13Suppl. 2, S241-250