safety and immunogenicity of subcutaneous hepatitis a vaccine in children with haemophilia

Safety and immunogenicity of subcutaneous hepatitis Avaccine in children with haemophilia

M. V. RAGNI,* J. M. LUSHER, M. A. KOERPER,à M. MANCO-JOHNSON± and D. S. KRAUSE**1

*Haemophilia Center of Western Pennsylvania, University of Pittsburgh Medical Center, Pittsburgh, PA; Haemophilia

Center, Children's Hospital of Michigan, Detroit, MI; àHaemophilia Center, University of California, San Francisco, CA;

±Mountain States Regional Haemophilia Center, Denver, CO; and **SmithKline Beecham Biologicals, Collegeville, PA, USA

HavrixÒ (hepatitis A vaccine, inactivated; Smith-Kline Beecham Biologicals, Collegeville, PA, USA3 ) iscurrently recommended for use in the prevention ofinfection with hepatitis A virus (HAV) [1], anonlipid enveloped4 RNA virus which accounts foras many as 50% of the cases of hepatitis per year[2]. Although transmission of HAV infection isprimarily via the faecal-oral route, transmissionmay also occur in individuals with haemophiliathrough clotting factor concentrate, particularlyproducts inactivated by solvent-detergent puri®ca-tion [3]. This puri®cation method has been ineffec-

tive against nonlipid enveloped viruses such asHAV, as evidenced by polymerase chain reaction(PCR) con®rmation of HAV in two of four batchesof factor VIII concentrate prepared by solvent-detergent methodology [4].

With a reported hepatitis antibody (anti-HAV)IgG seroprevalence of 40±70% in multitransfusedindividuals with haemophilia A or B [5±8], individu-als with haemophilia may be at risk for hepatitis Aand, thus, may bene®t from preventive immunizationwith hepatitis A vaccine. In addition, immunizationagainst hepatitis A may prevent potentially seriousliver disease in those already infected with hepatitisC. This vaccine is therefore recommended for indi-viduals with haemophilia and other congenitalbleeding disorders by The Medical and Scienti®cAdvisory Council of the National HaemophiliaFoundation [9]. The currently recommended routeof administration for the vaccine is intramuscular

Summary. Individuals with haemophilia are at riskfrom hepatitis A virus (HAV) infection throughexposure to blood products. HavrixÒ, an intramus-cular hepatitis A vaccine, is currently recommendedfor the prevention of disease caused by hepatitis Avirus. Because bleeding may complicate intramus-cular injections in those with bleeding disorders, weconducted a randomized, Phase IV clinical trial tocompare the safety and immunogenicity of HavrixÒ

given by the subcutaneous (s.c) vs. intramuscular(i.m.) route. A total of 45 children with haemo-philia were vaccinated subcutaneously, while their41 nonhaemophlic siblings were vaccinated intra-muscularly, at a dose of 720 Elisa units (EL.U.)2 attime 0 and 6 months. All children were anti-HAVand anti-HIV negative at baseline, and the haemo-philic group did not differ from their siblings inalanine aminotransferase (ALT; 25 IU L)1 vs. 22IU L)1), or in age; 8.5 years vs. 8.7 years. The

vaccine was well tolerated, with minor adverseevents being similar between groups; 21 (47%) vs.24 (58%), P > 0.05. Local symptoms includedsoreness in 39 (45%), erythema in 25 (29%),swelling in 21 (24%), and bruising in six (7%),with no differences between groups. The proportionseroconverting to anti-HAV IgG positive did notdiffer between groups; 98% vs. 97% at month 1;82% vs. 93% at month 6; and 100% vs. 100% atmonth 8, respectively. The HAV geometric meantitre was lower in those with haemophilia, 185 vs.233 mIU mL)1 at month 1; 68 vs. 94 mIU mL)1 atmonth 6; and 584 vs. 1082 mIU mL)1 at month 8,respectively. We conclude that HavrixÒ is safe andimmunogenic when administered s.c. in childrenwith haemophilia.

Keywords: anti-HAV, haemophilia, hepatitis A virus,immunogenicity, safety, vaccination.

Correspondence: Margaret V. Ragni, M.D., M.P.H., University ofPittsburgh School of Medicine, Haemophilia Center of WesternPennsylvania, 3636 Boulevard of the Allies, Pittsburgh, PA 15213±4306. Tel.: + 1 412 209 7288; fax: + 1 412 6834029

Accepted after revision 21 December 1999

Haemophilia (2000), 6, 98±103

98 Ó 2000 Blackwell Science Ltd

(i.m.), which may lead to bleeding or haematomaformation in individuals with congenital bleedingdisorders. There is, however, only limited experiencewith vaccines by the subcutaneous (s.c) route inindividuals with haemophilia [10±13]. We thereforeundertook a study of HavrixÒ administered by thes.c. route in children with haemophilia.

Materials and methods

Patient characteristics

This was a prospective, open-label, randomizedPhase IV clinical study to compare the safety andimmunogenicity of HavrixÒ, 720 Elisa units (EL.U.),administered at 0 and 6 months by s.c. injection inchildren with haemophilia aged between 2 and18 years old, with the same dose given by intramus-cular (i.m.) injection in their siblings. The age of thesiblings was within �5 years of that of the indexhaemophiliac. All subjects were HIV antibody neg-ative and anti-HAV negative at baseline. Patientswere enrolled at four haemophilia treatment centres,underwent a brief physical examination and labora-tory studies, including anti-HAV at baseline, at time0 (®rst vaccination), 1, 6, and 8 months. Reactoge-nicity was determined by assessment of signs andsymptoms at 1, 6, and 8 months. Personal diarieswere kept by all subjects or their parents of allsymptoms and medications throughout the study andwere reviewed in person by the physician and/ornurse at the 1, 6, and 8 month visits, and bytelephone when necessary.

Laboratory assays

Anti-HAV antibodies were measured by enzymeimmunoassay (EIA, Enzymun; Boehringer-Man-nheim, Indianapolis, IN, USA5 ), performed at theUniversity of Miami Hepatology Diagnostic Labora-tory. In this pseudo two-step sandwich assay, patientplasma is incubated with unlabelled hepatitis Aantigen in a liquid phase, and subsequently reactedwith two different monoclonal hepatitis A antibodiesto bind remaining unbound antigen. The ®rst anti-body is labelled with biotin and binds the antigen tothe surface of a streptavidin-coated tube; the secondantibody is labelled with peroxidase and allows thequanti®cation of the bound biotin-antibody-antigen-peroxidase-antibody complex [14]. This sensitiveassay can detect low-level circulating HAV antibodyin the range of 0±70 mIU mL)1 [14]. All samples werecoded and batch-tested, using one lot of monoclonalantibody per run.

Statistical analysis

Categoric data, including demographic data, adverseevents, and seroconversion rates, were statisticallyanalysed by v-square analysis, the Mantel-Haenszeltest, and Fisher's exact test. Continuous data,including age, baseline ALT, and anti-HAV geomet-ric mean titres, were analysed by Student's t-test.

Results

The patient demographics are listed in Table 1.A total of 86 children, 2±18 years of age, 45 withhaemophilia and 41 siblings without, were enrolledon the study. There were no differences in age,ethnicity, or baseline laboratory values between thegroups. There was a smaller proportion of females inthe group with haemophilia, consistent with theX-linked inheritance. Twelve (27%) of the haemo-philia patients were receiving factor concentrateprophylaxis to prevent haemorrhages, and two (4%)patients were receiving immune tolerance treatmentof antifactor VIII inhibitors. The vaccine was admin-istered subcutaneously or intramuscularly in the areaof the deltoid muscle of the upper arm in 69 (80%) orin the area of the vastus muscle of the thigh in 17(20%), with no difference between groups.

Safety

There were no serious adverse events. A total of 45(52%) children experienced at least one local minorevent after the ®rst and/or second immunization.There was no difference between groups; 21 (47%)children with haemophilia vs. 24 (58%) age-matchedsiblings, P > 0.05 (Table 2). The mean age of the 45children experiencing at least one minor relatedevent, 7.7 � 0.5 years, was younger than that of the41 children experiencing no minor related event;9.5 � 0.6 years, P < 0.05.

Soreness was the most common local nonseriousevent, occurring in 40%, followed by erythema in31%, swelling in 31%, and bruising in 9% of thesubjects, with no signi®cant difference in these eventsbetween haemophilic children and their nonhaemo-philic siblings. Local bruising and swelling, althoughexpected to be common among those with haemo-philia, was no more common than in their non-haemophilic siblings (Table 2).

Nonspeci®c symptoms were reported in a totalof 37 (43%) children and occurred less frequentlyin those with haemophilia, 15 (33%), than in theirnonhaemophilic siblings, 22 (54%), but this didnot reach statistical signi®cance (Table 2). The

SUBCUTANEOUS HEPATITIS A VACCINE IN HAEMOPHILIA 99

Ó 2000 Blackwell Science Ltd Haemophilia (2000), 6, 98±103

most common nonspeci®c symptoms were upperrespiratory symptoms in 29 (34%), headache in 10(12%), fever in four (5%), and gastrointestinalsymptoms in three (3%). Headaches occurred morecommonly in the nonhaemophilic siblings than inthe haemophiliacs, 19% vs. 4%, P � 0.004. Over-all, more nonspeci®c symptoms occurred after the®rst dose of the two-dose vaccination than thesecond (Table 2).

Immunogenicity

Two months following the second dose, all subjects inboth groups had seroconverted to anti-HAV IgGseropositivity, with no differences between groups inthe proportion seroconverting at month 1 and month 6(Table 3). Peak anti-HAV titres occurred at eightmonths, (2 months after the second immunization).Anti-HAV IgG geometric mean titres were signi®-cantly lower in haemophilic children as comparedwith their nonhaemophilic siblings at all time pointsfollowing immunization.

Discussion

Hepatitis A vaccine was found to be safe andimmunogenic in children with haemophilia when

administered by the subcutaneous route. Whencompared with their siblings who received thevaccine i.m., the haemophilic children who receivedthe vaccine s.c. experienced no greater frequency ofadverse events. Speci®cally, although local s.c. irri-tation is not uncommon with vaccines administeredby this route [13], the frequency of local soreness,swelling, and erythema was no greater in thehaemophilic children than in their nonhaemophilicsiblings.

The anti-HAV IgG titres peaked at eight months,with 100% seroconversion by 8 months, that is,2 months after the second dose. Among the haem-ophilic children, the HAV IgG geometric mean titreswere lower at each time point than in their non-haemophilic siblings. The potential reasons for thesedifferences in HAV antibody titre could relate to theroute of administration of the vaccine; the sensitivityof the antibody test employed; sex-related bodyweight differences; or immunological defects inhaemophilic children.

It is possible that the immunogenicity of a vaccinegiven by the subcutaneous route is inferior to thatgiven by the intramuscular route. For example,differences in s.c. processing of an immunogen orin the rapidity or extent to which an immunogenreaches the bloodstream could in¯uence the intensity

Table 1. Hepatitis A vaccine trial in haemophiliacs: Demographic information.

Haemophiliac* Sibling** Total*** P-value****

Mean age (yrs) 8.5 � 0.6 8.7 � 0.6 8.6 � 0.4 NS

Sex

Male 44 (98%) 19 (46%) 63 (73%)

Female 1 (2%) 22 (54%) 23 (27%) P < 0.05

Ethnicity

Caucasian 29 (64%) 34 (83%) 63 (73%)

Black 12 (27%) 5 (12%) 17 (20%)

Asian 2 (4%) 2 (5%) 4 (5%)

Hispanic 2 (4%) 0 (0%) 2 (2%) NS

Haemophilia Type

Haemophilia A 36 (80%) ± ±

Haemophilia B 9 (20%) ± ±

Haemophilia severity

Severe (< 0.01 U/mL) 26 (58%) ± ±

Moderate (0.01-.04 U/mL) 12 (27%) ± ±

Mild ( 0.05 U/mL) 7 (15%) ± ±

Haemophilia inhibitor present 4 (9%) ± ±

Baseline laboratory tests

Mean ALT (IU/mL) 25 � 2 22 � 1 24 � 1 NS

No. of baseline HIV Ab (+) 0/45 (0%) 0/41 (0%) 0/86 (0%)

No. of baseline HAV Ab (+) 0/45 (0%) 0/41 (0%) 0/86 (0%)

Vaccination site

Deltoid 36 (80%) 33 (80%) 69 (80%)

Thigh 9 (20%) 8 (20%) 17 (20%) NS

*n = 45; **n = 41; ***n = 86; ****P-value, based on comparison between haemophiliac and sibling.

100 M. V. RAGNI et al.

Haemophilia (2000), 6, 98±103 Ó 2000 Blackwell Science Ltd

of antibody response. Previous studies, however,suggest this is unlikely. Immunization of Italian,French, and American haemophilic children withhepatitis A vaccine [10±13] resulted in comparablesafety and seroconversion rates to those observed inthis study. As anti-HAV titres were not measured inthese studies, however, comparison of ef®cacy wasnot possible.

Another factor which may in¯uence anti-HAVantibody titres is the anti-HAV assay employed,

speci®cally the assay sensitivity and monoclonalantibodies utilized in detection of anti-HAV [14].Anti-HAV titres in this study were measured by avery sensitive assay that utilizes two differentmonoclonal antibodies to maximize antibodydetection [14]. Further, samples were run in batch,including all haemophilic and nonhaemophilicsamples in each run, and thus it is unlikely thatassay differences explain the antibody titre differ-ences.

Table 2 Hepatitis A vaccine trial in haemophiliacs: adverse events.

Month 0 Month 6 Total

Haemophiliacs* Siblings** Haemophiliacs* Siblings** Haemophilias* Siblings** P-value***

Minor local events

Soreness 15 13 14 15 18 21 P > 0.05

33% 62% 31% 36% 40% 51%

Erythema 9 2 9 7 14 11 P > 0.05

20% 5% 20% 17% 31% 27%

Swelling 8 5 9 3 14 7 P = 0.065

18% 12% 20% 7% 31% 17%

Bruising 2 2 2 0 4 2 P = 0.260

4% 5% 4% 0% 9% 5%

Subtotal 15 13 14 15 21 24 P > 0.05

33% 32% 31% 36% 47% 58%

Other events

Upper respiratory 11 15 2 3 12 17 P > 0.05

symptoms 24% 36% 4% 7% 27% 41%

Headache 2 7 0 4 2 8 P = 0.004

4% 17% 0% 10% 4% 19%

Fever 0 1 2 1 2 2 P = 0.285

0% 2% 4% 2% 4% 5%

Gastrointestinal 1 2 1 1 1 2 P = 0.360

symptoms 2% 5% 2% 2% 2% 5%

Other 5 4 5 6 8 10 P = 0.158

11% 10% 11% 15% 18% 24%

Subtotal 14 17 4 8 15 22 P > 0.05

31% 41% 9% 19% 33% 54%

*n = 45; **n = 41. ***P-value is based on comparison between haemophilic child and sibling.

Table 3. Immunogenicity of Hepatitis A vaccine in haemophiliacs.

Haemophiliacs* Siblings** P-value***

Anti-HAV Seroconversion Rate

Month 0 0/44 (0%) 0/41 (0%) ±

Month 1 44/45 (98%) 39/40 (97%) P = 0.504

Month 6 37/45 (82%) 38/41 (93%) P = 0.094

Month 8 45/45 (100%) 41/41 (100%) ±

Anti-HAV Geometric Mean Titre

Month 0 <33.00 <33.00 ±

Month 1 185.05 232.72 P < 0.01

Month 6 68.19 94.08 P < 0.01

Month 8 584.09 1082.06 P < 0.01

*Injected with HavrixÒ 720 EL.U. s.c.; n = 45. **Injected with HavrixÒ 720 EL.U. i.m.; n = 41. ***P-value is based on comparison

between haemophilic child and sibling.



Body weight may also account for differences inanti-HAV antibody titre between the groups. Theproportion of siblings who were female was signi®-cantly greater than the proportion of haemophiliacs,and as females are lighter on average than males, theymay have received a higher dose kg)1 of vaccine.

Immunologic defects present in the haemophilicchildren could explain their antibody titre differencescompared with their nonhaemophilic siblings. As allof the children in the present study, both haemophilicand nonhaemophilic, were anti-HIV negative, HIVimmunosuppression does not explain the antibodytitre differences observed. It is known that individu-als with haemophilia with chronic exposure toclotting factor concentrates have depressed immunesystem function, as measured by CD4 number,cytokine production, and monocyte function, in theabsence of HIV infection [15±18]. Although noprevious studies have compared haemophilic chil-dren with their nonhaemophilic siblings, studies ofanti-HIV negative haemophiliacs at this Center havefound reduced antibody responses to in¯uenza andpneumococcal vaccines in anti-HIV negative haem-ophilic subjects as compared with normal, nonhaem-ophilic subjects [15]. Whether reduced anti-HAVtitres following vaccination result in any less protec-tion is unknown but considered unlikely, as thereduced antibody responses observed with in¯uenzaand pneumococcal vaccines have not been associatedwith any lack of clinical protection. Moreover, anti-HAV titres obtained in this study were comparableto those reported in other studies of nonhaemophilicchildren [19, 20].

Thus, the ®ndings of this study con®rm thathepatitis A vaccine is safe and immunogenic whenadministered by the s.c. route in children withhaemophilia.

Acknowledgements

We thank Paula McGrath, R.N., Sue Ke¯er, R.N.,Sharon Eskern R.N., and Sheryl Giambartolomei,R.N. for coordination of the study in their haemo-philia centres; Ms. Maria Medina for coordination ofthe serologic assays,; Ms. Lynn Albright for coordi-nation of vaccine distribution; and Mrs. Karen Sabanfor secretarial support. We thank SmithKline Bee-cham for contributing the vaccine and anti-HAVassays. This study was supported by Grant No. U27/CCU31393 from the HHS Region III FederalHaemophilia Treatment Centers, and Grant No.ME97103 from the Maternal and Child HealthBureau, Pennsylvania State Department of Health,Harrisburg, PA, USA.

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safety and immunogenicity of subcutaneous hepatitis a vaccine in children with haemophilia

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