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Immunogenicity and tolerability of a virosome influenza vaccine compared to split influenza vaccine in patients with sickle cell anemia
Vaccine 28 (2010) 1117–1120
Contents lists available at ScienceDirect
Vaccine journal homepage: www.elsevier.com/locate/vaccine
Immunogenicity and tolerability of a virosome influenza vaccine compared to split influenza vaccine in patients with sickle cell anemia Alessandra R. Souza a,∗ , Josefina A.P. Braga a , Terezinha M. de Paiva b , Sandra R. Loggetto c , Raymundo S. Azevedo d , Lily Y. Weckx e a
Pediatric Hematology at the Federal University of São Paulo – Paulista School of Medicine, Brazil Section of Respiratory Virus at Adolfo Lutz Institute, Brazil Hematology Center, Brazil d Pathology Department of the School of Medicine of the University of São Paulo, Brazil e Pediatric Infectious Disease Discipline of the Federal University of São Paulo – Paulista School of Medicine, Brazil b c
a r t i c l e
i n f o
Article history: Received 22 July 2008 Received in revised form 4 May 2009 Accepted 14 May 2009
Keywords: Influenza vaccines Virosome vaccines Sickle cell anemia
a b s t r a c t The immunogenicity and tolerability of virosome and of split influenza vaccines in patients with sickle cell anemia (SS) were evaluated. Ninety SS patients from 8 to 34 years old were randomly assigned to receive either virosome (n = 43) or split vaccine (n = 47). Two blood samples were collected, one before and one 4–6 weeks after vaccination. Antibodies against viral strains (2006) A/New Caledonia (H1N1), A/California (H3N2), B/Malaysia were determined using the hemagglutinin inhibition test. Post-vaccine reactions were recorded over 7 days. Seroconversion rates for H1N1, H3N2 and B were 65.1%, 60.4% and 83.7% for virosome vaccine, and 68.0%, 61.7% and 68.0% for split vaccine. Seroprotection rates for H1N1, H3N2 e B were 100%, 97.6% and 69.7% for virosome, and 97.8%, 97.8% and 76.6% for split vaccine. No severe adverse reactions were recorded. Virosome and split vaccines in patients with sickle cell anemia were equally immunogenic, with high seroconversion and seroprotection rates. Both vaccines were well tolerated. © 2009 Elsevier Ltd. All rights reserved.
1. Introduction Sickle cell anemia (SS) is the most common monogenic inherited disorder in Brazil [1]. Due to functional asplenia and decrease in serum opsonizing activity, patients frequently develop severe infections by encapsulated organisms, especially pneumococcus [2,3]. Infection due to influenza virus leads to lesions in the epithelium of the respiratory tract, favouring secondary pneumococcal infection [4]. Moreover viral infection can also trigger sickle cell anemia crisis that may require hospital treatment [2,5]. Thus, an effective protection against influenza virus is necessary and vaccine is the main preventive measure [6]. Influenza vaccines most commonly used are inactivated split vaccine and subunit vaccines with purified viral antigens, hemagglutinin (HA) and neuraminidase (NA). The elderly, immunodeficient and immunologically naïve individuals such as children
∗ Corresponding author at: Rua Loefgren, 1998, 04040-003 São Paulo, SP, Brazil. Tel.: +55 11 5571 2944; fax: +55 11 5575 6928. E-mail addresses: [email protected] (A.R. Souza), josefi[email protected] (J.A.P. Braga), [email protected] (T.M. de Paiva), [email protected] (S.R. Loggetto), [email protected] (R.S. Azevedo), [email protected] (L.Y. Weckx). 0264-410X/$ – see front matter © 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2009.05.046
who were not previously exposed to influenza virus or vaccine present a decreased response to those vaccines [7,8]. Some studies have shown that the virosome influenza vaccine presents the same or even higher immunogenicity than conventional vaccines used in risk groups, such as the elderly [9–17]. The improvement in the immunogenicity could also benefit SS patients. The objectives of this study were to assess humoral immune response and tolerability to virosome influenza vaccine compared to split influenza vaccine in patients with sickle cell anemia. 2. Methods 2.1. Population The research was developed in the Reference Center for Special Immunobiologicals at the Federal University of São Paulo and at the Hematology Center, in São Paulo, Brazil, from May to November 2006. The project was approved by the Ethics Committee of both involved institutions. All participants or guardians gave written informed consent before study entry. Inclusion criteria were: 8 years or older, diagnosis of sickle cell anemia by hemoglobin electrophoresis and not having received influenza vaccine in 2006. Exclusion criteria were: fever
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A.R. Souza et al. / Vaccine 28 (2010) 1117–1120
Table 1 Demographic characteristics. Vaccine group
n
Median age in years (range)
Female gender (%)
Previous vaccination (%)
Virosomal Split
43 47
15 (8–33) 15 (8–34)
21 (48.8) 24 (51.1)
10 (23.0) 5 (10.6)
atimmunization, congenital or acquired immunodeficiency or use of immunosuppressors. Administration of blood products in the three months prior to or 14 days post-vaccination were also an exclusion criterion because immunoglobulins present in those components can interfere with immune response to vaccine. 2.2. Vaccines The influenza vaccines used in the study were single-dose inactivated virosome-adjuvanted Inflexal V® , lot #3000978, Berna Biotech Ltd., Switzerland, and multi-dose inactivated split vaccine, lot #0603056, Sanofi Pasteur, packed and controlled by Butantan Institute, São Paulo, Brazil. Each dose (0.5 mL) of the virosome or the split vaccine has 15 g of HA of each of the three strains of the influenza virus recommended by the WHO for the year 2006 in the Southern Hemisphere: A/New Caledonia/20/99(H1N1)-like, A/California/7/2004(H3N2)like and B/Malaysia/2506/2004-like. In the virosome vaccine, purified HA and NA are integrated to the lipid membrane of the virosome. This vaccine does not contain thimerosal as opposed to the split influenza vaccine which contains 2 g of thimerosal. Both vaccines are administered intramuscularly. Children under 9 years who had not been vaccined against influenza in the past received two doses of the vaccine (virosome or split) with the second dose after a month interval. 2.3. Randomization It was an unblinded study. SS patients were divided into two groups, by age and study entry: group I, individuals with sickle cell anemia (SS) receiving virosome vaccine; group II, SS individuals receiving split influenza vaccine.
or higher than 4-fold in HI antibody titers, and seroprotection as HI antibody titers higher than or equal to 1/40 [19].
2.5. Tolerability assessment Adverse events were registered in a diary during the 7 days after vaccination by the patients or parents/guardians. They were instructed to tick reactions and to note down any other event. A telephone call was made on the second and fifth day following the vaccination to check for any serious events and to solve any doubts. Intensity of reaction was graded subjectively, as mild (well tolerated symptoms), moderate (when symptoms interfered with some activities) and severe (when symptoms hindered every day activities). Any severe adverse event had to be reported throughout the study period.
2.6. Statistical analysis Assuming an 80% seroprotection [20], a sample of 100 patients (50 in each vaccine group) was calculated. The study would have a 70% power to demonstrate the equivalence between the virosome and split influenza vaccines, with a 20% acceptable difference between them and a 95% confidence interval. Statistical analysis was performed using MINITAB program, release 14.0. Mann–Whitney test was used to compare seroconversion between groups for each of the vaccine strains. Pearson’s Chi-square test was used to compare seroprotection rates between vaccines in the two groups. Adverse events reported by the two groups were compared using Chi-square test. Statistical significance was set at p < 0.05.
3. Results 2.4. Laboratory methods 3.1. Study population Blood samples were collected immediately before vaccination and 4–6 weeks afterwards (or 4–6 weeks after the second dose of the vaccine). Hemagglutinin antibodies against the three vaccine strains were assessed by hemagglutination inhibition test (HI) [18] at Adolfo Lutz Institute, São Paulo, Brazil. Results were expressed in geometric mean titers (GMT) of HI antibodies. Seroconversion was considered as an increase equal to
Ninety-eight patients with sickle cell anemia (SS) took part in the study. Eight SS patients were excluded, 4 of them because they had received blood transfusions post-vaccination and 4 because they failed to have the second blood sample collected. Patient characteristics are shown in Table 1. No significant differences between groups virosomal and split were observed regarding age, gender and previous vaccinations.
Table 2 Geometric mean titers, seroconversion and seroprotection rates after virosome or split vaccine in patients with sickle cell anemia. Vaccine group
Seroprotection rate pre-vaccination (%) N
H1N1
H3N2
GMT pre/post B
Virosomal
43
58.1
69.7
4.6
Split p
47
57.4
76.6
14.8
H1N1 40/320 40/320
H3N2 160/320 160/640
Seroconversion rate (%) B <10/40 10/40
Post-seroprotection rate (%)
H1N1
H3N2
B
H1N1
H3N2
B
65.1 (49.1; 79.0) 68.0 (52.9; 80.9) 0.519
60.4 (44.4; 75.0) 61.7 (46.4; 75.5) 0.79
83.7 (69.3; 93.2) 68.0 (52.9; 80.9) 0.399
100 (91.8; 100.0* ) 97.8 (88.7; 99.9) 0.999
97.6 (87.7; 99.9) 97.8 (88.7; 99.9) 0.999
69.7 (53.9; 82.8) 76,6 (59.7; 86.1) 0.485
H1N1: A/New Caledonia/20/99-like; H3N2: A/California/7/2004-like; B/Malaysia/2506/2004-like. GMT: mean geometric titers. Seroconversion: ≥4-fold increase in antibody HI titer. Seroprotection: HI titer ≥40. (;) 95% confidence interval. * 97.5% unilateral confidence interval.
A.R. Souza et al. / Vaccine 28 (2010) 1117–1120 Table 3 Incidence of local and systemic reactions after vaccination. Virosomal (N = 43)
Split (N = 47)
p (2 test)
Local reactions Pain Edema Induration Redness Itching Any local reaction
N (%) 18 (41.8) 6 (13.9) 7 (16.3) 3 (6.9) 1 (2.3) 20 (46.5)
N (%) 22 (46.8) 4 (8.5) 2 (4.2) 2 (4.2) 4 (8.5) 24 (51.0)
0.637 0.412 0.058 0.573 0.201 0.666
Systemic reactions Headache Myalgia Arthralgia Weakness Malaise Fever Backache Pharyngitis Earache Flu syndrome Rhinitis Abdominal pain Nausea Vomiting Diarrhoea Any systemic reaction
8 (18.6) 4 (9.3) 2 (4.6) 4 (9.3) 3 (6.9) 2 (4.6) 0 1 (2.3) 1 (2.3) 0 1 (2.3) 1 (2.3) 0 0 1 (2.3) 15 (34.8)
9 (19.1) 11 (23.4) 6 (12.7) 6 (12.7) 5 (10.6) 3 (6.3) 2 (4.2) 1 (2.1) 0 2 (4.2) 0 0 2 (4.2) 1 (2.1) 0 19 (40.4)
0.947 0.073 0.177 0.601 0.542 0.720 0.999 0.999 0.999 0.999 0.999 0.999 0.999 0.999 0.999 0.588
3.2. Immunogenicity Table 2 presents pre- and post-vaccine GMT results, seroconversion and pre- and post-seroprotection rates after virosome and split influenza vaccines in SS patients. No significant differences were noticed regarding the magnitude of the increase in geometric mean antibody titers, seroconversion or seroprotection rates for the three different strains. 3.3. Tolerability The frequency of local and systemic reactions is shown in Table 3. Local reactions occurred in 46.5% of cases after virosome vaccine and in 51.0%, after split vaccine. The most frequent local reactions were: localized pain, induration and edema. Systemic reactions were reported by 34.8% of individuals after virosome vaccine and by 40.4% of individuals after split vaccine, the most frequent being headache, myalgia and weakness. Most reactions were mild. Severe events were not reported. There were no significant differences between groups regarding adverse post-vaccine events. 4. Discussion In this study virosome influenza vaccine administered to individuals with sickle cell anemia (SS) proved to be as immunogenic and well tolerated as split influenza vaccine. The first virosomal vaccine produced for human use was the hepatitis A virosomal vaccine, licensed in 1996. After that came the influenza virosomal vaccine, licensed in 1997 [21]. Virosomes may be produced from a variety of enveloped viruses, including influenza virus, the resulting particles being similar to the original virus, except they lack the genetic material needed to replicate. Thus, functionally reconstituted influenza virosomes preserve the receptor-binding and membrane fusion activity of the viral hemagglutinin. These characteristics form the basis for their enhanced immunogenicity [21–24]. The split influenza vaccine possesses suboptimal antigen presentation [21]. There are few studies which have evaluated the immunogenicity of the split influenza vaccine in sickle cell anemia [20,25,26]. In one study, it was observed that SS adults with no
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functioning spleen showed lower levels of IgM post-split influenza vaccine [26]. There has been no consensus if SS patients present an adequate response to vaccination [27–31], probably due to functional asplenia. Impairment of spleen function in sickle cell anemia appears early, starting in the first year of life and following a progressive course due to recurrent sickling and microinfarctions in the spleen. Around the sixth to eighth year of life, this organ becomes fibrotic, a phenomenon known as autosplenectomy. In order to have homogeneous sample, we decided to include, in this study, patients with 8 years old and over [2,32,33]. Before vaccination, a great proportion of SS individuals presented protective antibodies titers against H1N1 and H3N2 strains (Table 2). This may be due mainly to previous natural infection, since we observed that most patients with sickle cell anemia (83.3%) were not routinely vaccinated against influenza despite recommendation by the Brazilian Health Ministry. However, antibody protective titers present before vaccination did not influence the response to influenza vaccines used which could be observed by the high seroconversion rate for the three influenza strains H1N1, H3N2 and B after vaccination in SS patients (Table 2). After the virosome vaccine seroconversion rates for the three viral strains (H1N1, H3N2, B) presented by SS patients ranged from 60.4% to 83.7% and seroprotection rates ranged from 69.7% to 100%. Similar results were found in the literature, with seroprotection rates reported using virosome vaccine ranging from 78.% to 95% in healthy children [10], from 90.4% to 93.8% in healthy adults and in some risk groups [11] and from 57% to 83% in HIV-infected children [17]. Some studies reported lower rates of local vaccine reactions associated with virosome vaccine as compared to conventional inactivated influenza vaccines [15,34]. However, no difference was observed between the two vaccines in respect to adverse events (Table 3). Mild local reactions were observed in 46.5% of virosome recipients and in 51.0% of split vaccine recipients (p = 0.666). The frequency of systemic events was also similar. In more recent studies, there is no difference in common systemic reactions like fever, headache, myalgia and arthralgia between recipients of influenza vaccine and placebo group [35]. This study had some limitations because many SS patients did not fulfill the eligibility criteria, led to a reduction in the power of the study which was to under 40%. Reduced differences would require a larger number of participants which was not possible due to the ineligibility of many SS patients. However, within the limitations of this study, and based on the results, it can be concluded that virosome and split influenza vaccine were immunogenic and well tolerated in SS patients. Conflict of interest There are no conflict of interest. Acknowledgments To Berna Biotech, Ltd. Laboratory, Switzerland and to Centers for Disease Control and Prevention, Atlanta, USA for the support in performing this research. Financing source: This project was partially sponsored by Berna Biotech Laboratory, Switzerland and Centers for Disease Control and Prevention (CDC), Atlanta, USA. References [1] Zago MA. Considerac¸ões gerais. In: Manual de diagnóstico e tratamento das doenc¸as falciformes. Brasília: ANVISA; 2001. p. 7–12.
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[2] Serjeant GR, Serjeant BE. Sickle cell disease. 3rd ed. Oxford: University Press; 2001. p. 153–166. [3] Dover GJ, Platt OS. Sickle cell disease. In: Nathan DG, Orkin SH, editors. Hematology of infancy and childhood. 5th ed. Philadelphia: W.B. Saunders Company; 1998. p. 762–809. [4] Glezen WP. Influenza viruses. In: Feigin RD, Cherry JD, editors. Textbook of pediatric infectious diseases. 4th ed. Philadelphia: WB Saunders Co.; 1998. p. 2024–41. [5] Paterson JCS, Sprague CC. Observation on the genesis of crises in sickle cell anemia. Am J Med 1959;50:1502. [6] Greenberg HB, Piedra PA. Immunization against viral respiratory disease: a review. Pediatr Infect Dis J 2004;23:S254–61. [7] Zanetti AR, Amendola A, Besana S, Boschini A, Tanzi E. Safety and immunogenicity of influenza vaccination in individuals infected with HIV. Vaccine 2002;20:B29–32. [8] Nichol KL. The efficacy, effectiveness and cost-effectiveness of inactivated influenza virus vaccine. Vaccine 2003;21:1769–75. [9] Herzog C, Metcalfe IC, Schaad UB. Virosome influenza vaccine in children. Vaccine 2002;20:B24–8. [10] Kanra G, Marchisio P, Feiterna-Sperling C, Gaedicke G, Lazar H, Durrer P, et al. Comparison of immunogenicity and tolerability of a virosome-adjuvanted and a split influenza vaccine in children. Pediatr Infect Dis J 2004;23:300–6. [11] De Brujin IA, Nauta J, Gerez L, Palache AM. Virosomal influenza vaccine: a safe and effective influenza vaccine with high efficacy in elderly and subjects with low pre-vaccination antibody titers. Virus Res 2004;103(1–2):139–45. [12] Glück R, Mischler R, Finkel B, Que JU, Scarpa B, Cryz Jr SJ. Immunogenicity of new virosome vaccine in elderly people. Lancet 1994;344:160–3. [13] Conne P, Gauthey L, Vernet P, Althaus B, Que JU, Finkel B, et al. Immunogenicity of trivalent subunit versus virosome formulated influenza vaccines in geriatric patients. Vaccine 1997;15(15):1675–9. [14] Schaad UB, Bühlmann U, Burger R, Ruedeberg A, Wilder-Smith A, Rutishauser M, et al. Comparison of immunogenicity and safety of a virosome influenza vaccine with those of a subunit influenza vaccine in pediatric patients with cystic fibrosis. Antimicrob Agents Chemother 2000;44(5):1163–7. [15] Pregliasco F, Mensi C, Serpilli W, Speccher L, Masella P, Belloni A. Immunogenicity and safety of three commercial influenza vaccines in institutionalized elderly. Aging (Milano) 2001;13(1):38–43. [16] Ruf BR, Colberg K, Frick M, Preusche A. Open, randomized study to compare the immunogenicity and reactogenicity of an influenza split vaccine with an MF 59-adjuvanted subunit vaccine and a virosome-based subunit vaccine in elderly. Infection 2004;32(4):191–8. [17] Zuccotti GV, Zenga A, Durando P, Massone L, Bruzzone B, Sala D, et al. Immunogenicity and tolerability of a trivalent virosomal influenza vaccine in a cohort of HIV-infected children. J Intern Med Res 2004;32:492–9. [18] Palmer DF, Coleman MT, Dowdle WR, Schild GC. Hemagglutination–inhibition test. In: Welfare PHS, editor. Advanced laboratory techniques for influenza diagnosis. Atlanta: U.S. Department of Health; 1975. p. 25–62.
[19] The European Agency for the Evaluation of Medicinal Products (EMEA); Committee for Proprietary Medicinal Products (CPMP). Note for guidance on harmonization of requirements for influenza vaccine (CPMP/BWP/214/96), London, March 12, 1997. [20] Glezen WP, Glezen LS, Alcorn R. Trivalent, inactivated influenza virus vaccine in children with sickle cell disease. Am J Dis Child 1983;137:1095–7. [21] Mischler R, Metcalfe IC. Inflexal® V a trivalent virosome subunit influenza vaccine: production. Vaccine 2002;20:B17–23. [22] Glück R, Metcalfe IC. New technology platforms in the development of vaccines for the future. Vaccine 2002;20:B10–6. [23] Huckriede A, Bungener L, Stegmann T, Daemen T, Medema J, Palache AM, et al. The virosome concept for influenza vaccines. Vaccine 2005;23S. S1/26–S1/38. [24] Weniger BG, Papania MJ. Alternative vaccine delivery methods. In: Plotkin AS, Orenstein WA, Offit PA, editors. Vaccine. 5th ed. Philadelphia: W.B. Saunders; 2008. p. 1357–92. [25] Steinberg E, Overturf GD, Portnoy B, Powars DR, Boyer KM, Cherry JD. Serologic and clinical response of children with sickle cell disease to bivalent influenza A split virus vaccine. J Pediatr 1978;92(5):823–5. [26] Ballester OF, Abdallah JM, Prasad A. Impaired IgM antibody responses to an influenza virus vaccine in adults with sickle cell anemia. Am J Hematol 1985;20:409–12. [27] Ahonkhai VI, Landesman SH, Fikrig SM, Schmalzer EA, Brown AK, Cherubin CE. Failure of pneumococcal vaccine in children with sickle cell disease. N Engl J Med 1979;310(1):26–7. [28] Bjornson AB, Falletta JM, Verter JI, Buchanan GR, Miller ST, Pegelow CH, et al. Serotype-specific immunoglobulin G antibody responses to pneumococcal polysaccharide vaccine in children with sickle cell anemia: effects of continued penicillin prophylaxis. J Pediatr 1996;129(6):828–35. [29] Frank AL, Labotta RJ, Rao S, Frisone LR, McVerry PH, Samuelson JS, et al. Haemophilus influenzae tipo b immunization of children with sickle cell diseases. Pediatrics 1988;82(4):571–5. [30] Rubin LG, Voulalas D, Carmody L. Immunization of children with sickle cell disease with Haemophilus influenzae type b polysaccharide vaccine. Pediatrics 1989;84(3):509–13. [31] Hord J, Windsor B, Koehler M, Blatt J, Janosky J, Mirro J. Diminished antibody response to hepatitis B immunization in children with sickle cell disease. J Pediatr Hematol Oncol 2002;24(7):548–9. [32] Groto HZW, Costa FF. Pattern of splenic phagocytic function in Brazilian patients with sickle cell disease. Rev Paul Med 1992;110(6):262–6. [33] Lane PA. Sickle cell disease. Pediatr Clin North Am 1996;43(3):639–64. [34] De Bruijn IA, Nauta J, Gerez L, Palache AM. The virosomal influenza vaccine Invivac® : immunogenicity and tolerability compared to an adjuvanted influenza vaccine (Fluad® ) in elderly subjects. Vaccine 2006;24:6629– 31. [35] Center for Disease Control and Prevention and Control of Influenza: recomendations of the Advisory Committee on Immunization Practices (ACIP). Morbid Mortal Weekly Rep 2006;55:1–42.
Contents lists available at ScienceDirect
Vaccine journal homepage: www.elsevier.com/locate/vaccine
Immunogenicity and tolerability of a virosome influenza vaccine compared to split influenza vaccine in patients with sickle cell anemia Alessandra R. Souza a,∗ , Josefina A.P. Braga a , Terezinha M. de Paiva b , Sandra R. Loggetto c , Raymundo S. Azevedo d , Lily Y. Weckx e a
Pediatric Hematology at the Federal University of São Paulo – Paulista School of Medicine, Brazil Section of Respiratory Virus at Adolfo Lutz Institute, Brazil Hematology Center, Brazil d Pathology Department of the School of Medicine of the University of São Paulo, Brazil e Pediatric Infectious Disease Discipline of the Federal University of São Paulo – Paulista School of Medicine, Brazil b c
a r t i c l e
i n f o
Article history: Received 22 July 2008 Received in revised form 4 May 2009 Accepted 14 May 2009
Keywords: Influenza vaccines Virosome vaccines Sickle cell anemia
a b s t r a c t The immunogenicity and tolerability of virosome and of split influenza vaccines in patients with sickle cell anemia (SS) were evaluated. Ninety SS patients from 8 to 34 years old were randomly assigned to receive either virosome (n = 43) or split vaccine (n = 47). Two blood samples were collected, one before and one 4–6 weeks after vaccination. Antibodies against viral strains (2006) A/New Caledonia (H1N1), A/California (H3N2), B/Malaysia were determined using the hemagglutinin inhibition test. Post-vaccine reactions were recorded over 7 days. Seroconversion rates for H1N1, H3N2 and B were 65.1%, 60.4% and 83.7% for virosome vaccine, and 68.0%, 61.7% and 68.0% for split vaccine. Seroprotection rates for H1N1, H3N2 e B were 100%, 97.6% and 69.7% for virosome, and 97.8%, 97.8% and 76.6% for split vaccine. No severe adverse reactions were recorded. Virosome and split vaccines in patients with sickle cell anemia were equally immunogenic, with high seroconversion and seroprotection rates. Both vaccines were well tolerated. © 2009 Elsevier Ltd. All rights reserved.
1. Introduction Sickle cell anemia (SS) is the most common monogenic inherited disorder in Brazil [1]. Due to functional asplenia and decrease in serum opsonizing activity, patients frequently develop severe infections by encapsulated organisms, especially pneumococcus [2,3]. Infection due to influenza virus leads to lesions in the epithelium of the respiratory tract, favouring secondary pneumococcal infection [4]. Moreover viral infection can also trigger sickle cell anemia crisis that may require hospital treatment [2,5]. Thus, an effective protection against influenza virus is necessary and vaccine is the main preventive measure [6]. Influenza vaccines most commonly used are inactivated split vaccine and subunit vaccines with purified viral antigens, hemagglutinin (HA) and neuraminidase (NA). The elderly, immunodeficient and immunologically naïve individuals such as children
∗ Corresponding author at: Rua Loefgren, 1998, 04040-003 São Paulo, SP, Brazil. Tel.: +55 11 5571 2944; fax: +55 11 5575 6928. E-mail addresses: [email protected] (A.R. Souza), josefi[email protected] (J.A.P. Braga), [email protected] (T.M. de Paiva), [email protected] (S.R. Loggetto), [email protected] (R.S. Azevedo), [email protected] (L.Y. Weckx). 0264-410X/$ – see front matter © 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2009.05.046
who were not previously exposed to influenza virus or vaccine present a decreased response to those vaccines [7,8]. Some studies have shown that the virosome influenza vaccine presents the same or even higher immunogenicity than conventional vaccines used in risk groups, such as the elderly [9–17]. The improvement in the immunogenicity could also benefit SS patients. The objectives of this study were to assess humoral immune response and tolerability to virosome influenza vaccine compared to split influenza vaccine in patients with sickle cell anemia. 2. Methods 2.1. Population The research was developed in the Reference Center for Special Immunobiologicals at the Federal University of São Paulo and at the Hematology Center, in São Paulo, Brazil, from May to November 2006. The project was approved by the Ethics Committee of both involved institutions. All participants or guardians gave written informed consent before study entry. Inclusion criteria were: 8 years or older, diagnosis of sickle cell anemia by hemoglobin electrophoresis and not having received influenza vaccine in 2006. Exclusion criteria were: fever
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A.R. Souza et al. / Vaccine 28 (2010) 1117–1120
Table 1 Demographic characteristics. Vaccine group
n
Median age in years (range)
Female gender (%)
Previous vaccination (%)
Virosomal Split
43 47
15 (8–33) 15 (8–34)
21 (48.8) 24 (51.1)
10 (23.0) 5 (10.6)
atimmunization, congenital or acquired immunodeficiency or use of immunosuppressors. Administration of blood products in the three months prior to or 14 days post-vaccination were also an exclusion criterion because immunoglobulins present in those components can interfere with immune response to vaccine. 2.2. Vaccines The influenza vaccines used in the study were single-dose inactivated virosome-adjuvanted Inflexal V® , lot #3000978, Berna Biotech Ltd., Switzerland, and multi-dose inactivated split vaccine, lot #0603056, Sanofi Pasteur, packed and controlled by Butantan Institute, São Paulo, Brazil. Each dose (0.5 mL) of the virosome or the split vaccine has 15 g of HA of each of the three strains of the influenza virus recommended by the WHO for the year 2006 in the Southern Hemisphere: A/New Caledonia/20/99(H1N1)-like, A/California/7/2004(H3N2)like and B/Malaysia/2506/2004-like. In the virosome vaccine, purified HA and NA are integrated to the lipid membrane of the virosome. This vaccine does not contain thimerosal as opposed to the split influenza vaccine which contains 2 g of thimerosal. Both vaccines are administered intramuscularly. Children under 9 years who had not been vaccined against influenza in the past received two doses of the vaccine (virosome or split) with the second dose after a month interval. 2.3. Randomization It was an unblinded study. SS patients were divided into two groups, by age and study entry: group I, individuals with sickle cell anemia (SS) receiving virosome vaccine; group II, SS individuals receiving split influenza vaccine.
or higher than 4-fold in HI antibody titers, and seroprotection as HI antibody titers higher than or equal to 1/40 [19].
2.5. Tolerability assessment Adverse events were registered in a diary during the 7 days after vaccination by the patients or parents/guardians. They were instructed to tick reactions and to note down any other event. A telephone call was made on the second and fifth day following the vaccination to check for any serious events and to solve any doubts. Intensity of reaction was graded subjectively, as mild (well tolerated symptoms), moderate (when symptoms interfered with some activities) and severe (when symptoms hindered every day activities). Any severe adverse event had to be reported throughout the study period.
2.6. Statistical analysis Assuming an 80% seroprotection [20], a sample of 100 patients (50 in each vaccine group) was calculated. The study would have a 70% power to demonstrate the equivalence between the virosome and split influenza vaccines, with a 20% acceptable difference between them and a 95% confidence interval. Statistical analysis was performed using MINITAB program, release 14.0. Mann–Whitney test was used to compare seroconversion between groups for each of the vaccine strains. Pearson’s Chi-square test was used to compare seroprotection rates between vaccines in the two groups. Adverse events reported by the two groups were compared using Chi-square test. Statistical significance was set at p < 0.05.
3. Results 2.4. Laboratory methods 3.1. Study population Blood samples were collected immediately before vaccination and 4–6 weeks afterwards (or 4–6 weeks after the second dose of the vaccine). Hemagglutinin antibodies against the three vaccine strains were assessed by hemagglutination inhibition test (HI) [18] at Adolfo Lutz Institute, São Paulo, Brazil. Results were expressed in geometric mean titers (GMT) of HI antibodies. Seroconversion was considered as an increase equal to
Ninety-eight patients with sickle cell anemia (SS) took part in the study. Eight SS patients were excluded, 4 of them because they had received blood transfusions post-vaccination and 4 because they failed to have the second blood sample collected. Patient characteristics are shown in Table 1. No significant differences between groups virosomal and split were observed regarding age, gender and previous vaccinations.
Table 2 Geometric mean titers, seroconversion and seroprotection rates after virosome or split vaccine in patients with sickle cell anemia. Vaccine group
Seroprotection rate pre-vaccination (%) N
H1N1
H3N2
GMT pre/post B
Virosomal
43
58.1
69.7
4.6
Split p
47
57.4
76.6
14.8
H1N1 40/320 40/320
H3N2 160/320 160/640
Seroconversion rate (%) B <10/40 10/40
Post-seroprotection rate (%)
H1N1
H3N2
B
H1N1
H3N2
B
65.1 (49.1; 79.0) 68.0 (52.9; 80.9) 0.519
60.4 (44.4; 75.0) 61.7 (46.4; 75.5) 0.79
83.7 (69.3; 93.2) 68.0 (52.9; 80.9) 0.399
100 (91.8; 100.0* ) 97.8 (88.7; 99.9) 0.999
97.6 (87.7; 99.9) 97.8 (88.7; 99.9) 0.999
69.7 (53.9; 82.8) 76,6 (59.7; 86.1) 0.485
H1N1: A/New Caledonia/20/99-like; H3N2: A/California/7/2004-like; B/Malaysia/2506/2004-like. GMT: mean geometric titers. Seroconversion: ≥4-fold increase in antibody HI titer. Seroprotection: HI titer ≥40. (;) 95% confidence interval. * 97.5% unilateral confidence interval.
A.R. Souza et al. / Vaccine 28 (2010) 1117–1120 Table 3 Incidence of local and systemic reactions after vaccination. Virosomal (N = 43)
Split (N = 47)
p (2 test)
Local reactions Pain Edema Induration Redness Itching Any local reaction
N (%) 18 (41.8) 6 (13.9) 7 (16.3) 3 (6.9) 1 (2.3) 20 (46.5)
N (%) 22 (46.8) 4 (8.5) 2 (4.2) 2 (4.2) 4 (8.5) 24 (51.0)
0.637 0.412 0.058 0.573 0.201 0.666
Systemic reactions Headache Myalgia Arthralgia Weakness Malaise Fever Backache Pharyngitis Earache Flu syndrome Rhinitis Abdominal pain Nausea Vomiting Diarrhoea Any systemic reaction
8 (18.6) 4 (9.3) 2 (4.6) 4 (9.3) 3 (6.9) 2 (4.6) 0 1 (2.3) 1 (2.3) 0 1 (2.3) 1 (2.3) 0 0 1 (2.3) 15 (34.8)
9 (19.1) 11 (23.4) 6 (12.7) 6 (12.7) 5 (10.6) 3 (6.3) 2 (4.2) 1 (2.1) 0 2 (4.2) 0 0 2 (4.2) 1 (2.1) 0 19 (40.4)
0.947 0.073 0.177 0.601 0.542 0.720 0.999 0.999 0.999 0.999 0.999 0.999 0.999 0.999 0.999 0.588
3.2. Immunogenicity Table 2 presents pre- and post-vaccine GMT results, seroconversion and pre- and post-seroprotection rates after virosome and split influenza vaccines in SS patients. No significant differences were noticed regarding the magnitude of the increase in geometric mean antibody titers, seroconversion or seroprotection rates for the three different strains. 3.3. Tolerability The frequency of local and systemic reactions is shown in Table 3. Local reactions occurred in 46.5% of cases after virosome vaccine and in 51.0%, after split vaccine. The most frequent local reactions were: localized pain, induration and edema. Systemic reactions were reported by 34.8% of individuals after virosome vaccine and by 40.4% of individuals after split vaccine, the most frequent being headache, myalgia and weakness. Most reactions were mild. Severe events were not reported. There were no significant differences between groups regarding adverse post-vaccine events. 4. Discussion In this study virosome influenza vaccine administered to individuals with sickle cell anemia (SS) proved to be as immunogenic and well tolerated as split influenza vaccine. The first virosomal vaccine produced for human use was the hepatitis A virosomal vaccine, licensed in 1996. After that came the influenza virosomal vaccine, licensed in 1997 [21]. Virosomes may be produced from a variety of enveloped viruses, including influenza virus, the resulting particles being similar to the original virus, except they lack the genetic material needed to replicate. Thus, functionally reconstituted influenza virosomes preserve the receptor-binding and membrane fusion activity of the viral hemagglutinin. These characteristics form the basis for their enhanced immunogenicity [21–24]. The split influenza vaccine possesses suboptimal antigen presentation [21]. There are few studies which have evaluated the immunogenicity of the split influenza vaccine in sickle cell anemia [20,25,26]. In one study, it was observed that SS adults with no
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functioning spleen showed lower levels of IgM post-split influenza vaccine [26]. There has been no consensus if SS patients present an adequate response to vaccination [27–31], probably due to functional asplenia. Impairment of spleen function in sickle cell anemia appears early, starting in the first year of life and following a progressive course due to recurrent sickling and microinfarctions in the spleen. Around the sixth to eighth year of life, this organ becomes fibrotic, a phenomenon known as autosplenectomy. In order to have homogeneous sample, we decided to include, in this study, patients with 8 years old and over [2,32,33]. Before vaccination, a great proportion of SS individuals presented protective antibodies titers against H1N1 and H3N2 strains (Table 2). This may be due mainly to previous natural infection, since we observed that most patients with sickle cell anemia (83.3%) were not routinely vaccinated against influenza despite recommendation by the Brazilian Health Ministry. However, antibody protective titers present before vaccination did not influence the response to influenza vaccines used which could be observed by the high seroconversion rate for the three influenza strains H1N1, H3N2 and B after vaccination in SS patients (Table 2). After the virosome vaccine seroconversion rates for the three viral strains (H1N1, H3N2, B) presented by SS patients ranged from 60.4% to 83.7% and seroprotection rates ranged from 69.7% to 100%. Similar results were found in the literature, with seroprotection rates reported using virosome vaccine ranging from 78.% to 95% in healthy children [10], from 90.4% to 93.8% in healthy adults and in some risk groups [11] and from 57% to 83% in HIV-infected children [17]. Some studies reported lower rates of local vaccine reactions associated with virosome vaccine as compared to conventional inactivated influenza vaccines [15,34]. However, no difference was observed between the two vaccines in respect to adverse events (Table 3). Mild local reactions were observed in 46.5% of virosome recipients and in 51.0% of split vaccine recipients (p = 0.666). The frequency of systemic events was also similar. In more recent studies, there is no difference in common systemic reactions like fever, headache, myalgia and arthralgia between recipients of influenza vaccine and placebo group [35]. This study had some limitations because many SS patients did not fulfill the eligibility criteria, led to a reduction in the power of the study which was to under 40%. Reduced differences would require a larger number of participants which was not possible due to the ineligibility of many SS patients. However, within the limitations of this study, and based on the results, it can be concluded that virosome and split influenza vaccine were immunogenic and well tolerated in SS patients. Conflict of interest There are no conflict of interest. Acknowledgments To Berna Biotech, Ltd. Laboratory, Switzerland and to Centers for Disease Control and Prevention, Atlanta, USA for the support in performing this research. Financing source: This project was partially sponsored by Berna Biotech Laboratory, Switzerland and Centers for Disease Control and Prevention (CDC), Atlanta, USA. References [1] Zago MA. Considerac¸ões gerais. In: Manual de diagnóstico e tratamento das doenc¸as falciformes. Brasília: ANVISA; 2001. p. 7–12.
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