Low rate of seroconversion after vaccination with a split virion, adjuvanted pandemic H1N1 influenza vaccine in HIV-1-infected patients
Bickel, Markusa; Wieters, Imkea; Khaykin, Pavela; Nisius, Gabia; Haberl, Annettea; Stephan, Christopha; Von Hentig, Nilsa; Herrmann, Evac; Doerr, Hans Wb; Brodt, Hans Ra; Allwinn, Reginab
bInstitute of Medical Virology, Germany
cInstitute of Biostatistics and Mathematical Modeling, Clinic of the Goethe University, Frankfurt, Germany.
Received 12 January, 2010
Revised 1 March, 2010
Accepted 8 March, 2010
Correspondence to Dr Markus Bickel, HIVCENTER, Goethe University Clinic, Frankfurt, Germany. Tel: +49 69 6301 7478; fax: +49 69 6301 84325; e-mail: firstname.lastname@example.org
Objective: To determine rates of seroconversion after single vaccination with a novel split virion, inactivated, adjuvanted pandemic H1N1 influenza vaccine (A/California/7/2009) in HIV-1-infected patients (ClinicalTrials.gov Identifier: NCT01017172).
Design: Single center diagnostic study.
Setting: Institutional HIV outpatient department of an urban university clinic.
Participants: Adult HIV-1-infected individuals.
Intervention: Serum samples were taken before and 21 days after vaccination.
Main outcome measures: Antibody titers determined by hemagglutination inhibition assay. Seroconversion to vaccination was defined by either an antibody titer of 1: 10 or less before and of at least 1: 40 after or at least 1: 10 before and at least four-fold increase in antibody titer 21 days after single vaccination.
Results: One hundred and sixty patients (125 men/35 women) were analyzed. Before vaccination, 23 patients (14.4%) had a hemagglutination inhibition assay titer of at least 1: 40. A median of 22 ± 3 days after vaccination, 110 (69%) patients seroconverted. Seroconverters were younger (45.1 ± 10.0 vs. 48.8 ± 11.3 years; P = 0.04), had a higher CD4 cell count (532 ± 227 vs. 475 ± 281 cells/μl; P = 0.03) and were more likely to have received a previous H5N1 vaccination in 2009 (25 vs. 8%; P = 0.02) when compared to nonresponders. No other significant differences were found comparing the two groups (prevaccination hemagglutination inhibition assay titer of ≥1: 40, AIDS, HAART, HIV RNA PCR <50 copies/ml or CD4 nadir, CD4 and CD8 percentage, sex, BMI, chronic hepatitis B or C).
Conclusion: Seroconversion after one dose of a split virion, inactivated, adjuvanted pandemic H1N1 influenza vaccine of HIV-infected patients was 69%. Studies to investigate whether a second dose of the vaccine will increase seroconversion rate are needed.
In September 2009, a novel split virion, inactivated, adjuvanted pandemic H1N1 influenza vaccine (A/California/7/2009 NYMC X-179A; Pandemrix; Glaxo Smith Kline Dresden, Dresden, Germany) was licensed in most European countries. The Center for Disease Control as well as the German Health Agency recommends vaccination for immunocompromised patients because these patients are likely to be at a higher risk of a H1N1 infection and a more severe course of disease [1,2]. Although recommended, the immunogenicity of this vaccine in this patient population is yet unknown. Protection against influenza is mediated by virus-specific antibodies (Abs) and depends on the humoral immune response [3,4], which might be impaired in HIV-infected individuals . Several studies investigating protective Ab response of HIV-1-infected patients after seasonal influenza vaccination have shown lower response rates, especially in patients with lower CD4 cell counts, previous AIDS and without HAART as compared to uninfected persons [6–13]. In three studies on HIV-infected patients [8,14,15], it was shown that adjuvanted seasonal influenza vaccines resulted in similar or higher rates of immunoresponse when compared to nonadjuvanted counterparts.
Therefore, our aim was to investigate the rate of seroconversion after vaccination with this novel H1N1 vaccine. This report is a preliminary analysis of an ongoing study (ClinicalTrials.gov Identifier: NCT01017172).
Adult patients from the HIVCENTER, Department of Infectious Disease at the Goethe University Clinic, who were routinely scheduled to receive a H1N1 vaccination, were asked to participate in this diagnostic study. The study protocol was approved by the local ethics committee on 11 November 2009. Patients older than 18 years, with known HIV-1 infection who gave written informed consent were able to participate in this study. Once agreed, a 10 ml blood sample was taken immediately before and 21 days after the first vaccination, centrifuged (10 min at 1500 rpm) and sera were frozen (−20°C) for further analysis. All clinical and HIV-related data were retrieved from the charts and had to be less than 3-month-old for the analysis.
Split influenza virus, inactivated, containing 3.75 μg antigen equivalent to A/California/7/2009 (H1N1)v-like strain (X-179A) hemagglutinin with AS03 adjuvant composed of squalene (10.69 mg), DL-α-tocopherol (11.86 mg) and polysorbate 80 (4.86 mg; Pandemrix; GSK Dresden), was used for a single intramuscular vaccination into the deltoid muscle.
Hemagglutination inhibition test
To investigate immunity, a hemagglutination inhibition (HAI) assay was done after removing naturally occurring, nonspecific inhibitors from the sera, according to the WHO guidance . Briefly, a standardized quantity of hemagglutinin antigen was mixed with serially diluted antisera, and red blood cells were added to determine specific binding of Ab to the hemagglutinin molecule. Reagents used for each testing were standardized fresh red blood cells (RBCs) of turkeys in Alsever's solution (Bundesinstitut für Risikobewertung, Alt-Marienfelde, Berlin, Germany) and H1N1-Virus split antigen (A/California/7/2009 NYMC X-179A; Pandemrix; GSK Dresden). Titers below the detection limit of 1: 10 were assigned to a value of 1: 5 for the purpose of calculating the geometric mean titer. According to European and International guidance, seroconversion after vaccination was defined by either an H1N1 Ab titer of 1: 10 or less before and of at least 1: 40 after or at least 1: 10 before and at least four-fold increase in Ab titer 21 days after vaccination [17,18].
For a comparison between the groups, the geometric mean Ab titers with the corresponding 95% confidence interval (CI) were calculated. Other values are shown as mean values ± SD. Continuous variables were compared using the nonparametric Man–Whitney test; nominal values were compared using Fisher's Exact Test or the χ2 test. All tests were two-tailed for a significance level of 0.05. Statistical analyses were done using SPSS for Windows, release 16 (SPSS Inc., Chicago, Illinois, USA).
Up to now 160 patients (125 men/35 women) completed the study and were evaluated. Before vaccination, 23 of 160 patients (14.4%) had a HAI titer of 1: 40 or more. A median of 22 ± 3 days after vaccination, 120 of 160 patients (75%) had a HAI titer of 1: 40 or more. Seroconversion was found in 110 of 160 patients (69%). The geometric mean HAI titer after vaccination for those who seroconverted was 205.9 (95% CI 169.0–250.1) and 16.9 (95% CI 11.6–24.8) for those who did not (P < 0.0001). Table 1 summarizes the clinical and laboratory characteristics. Patients who seroconverted were younger (45.1 ± 10.0 vs. 48.8 ± 11.3 years; P = 0.04), had a higher absolute CD4 cell count (532 ± 227 vs. 475 ± 281 cells/μl; P = 0.03) and were more likely to have received a previous H5N1 vaccination in 2009 (25 vs. 8%; P = 0.02) when compared to nonresponders. The proportion of patients with a HAI titer of at least 1: 40 before vaccination was not different when comparing those who previously had a H5N1 vaccination in 2009 (10%) with those who did not (16%; P = 0.39). No significant differences were found comparing the two groups for prevaccination HAI titer of at least 1: 40 or its geometric mean, for AIDS, HAART, HIV RNA PCR less than 50 copies/ml or CD4 nadir, CD4 and CD8 percentage, sex, BMI and previous seasonal influenza vaccination in 2009. There was also no significant difference between patients with a chronic hepatitis B, C or both (data not shown).
Based on data of the seasonal influenza, a H1N1 virus-specific Ab titer of at least 1: 40 is currently assumed to confer protective immunity against the H1N1 influenza virus, which was found in 75% 3 weeks after a single immunization. The rate of seroconversion in our study was 69% (95% CI 61.0–75.8) and, therefore, lower when compared to results reported from the general population of three trials using the same vaccine (Table 2) [http://www.gsk-clinicalstudyregister.com/quick-search-list.jsp?tab=results&letterrange=All&type=Compound&item=H1n1+Pandemic+Influenza+Vaccine&studyType=All&phase=All&status=All&population=All&marketing=All&country=All&studyId= (Last accessed 05 January 2010)]. The HIV-infected patients in our study were older than the participants included in the studies of the general population, which may in part explain the lower rates of seroconversion. On the other hand, when comparing results of patients who were of 60 years or older, the rate of seroconversion of the HIV-infected patients was less than half as compared to results from a study on the general population, which was stratified for age (Table 2). Older individuals in general had a lower response to various H1N1 as well as seasonal influenza vaccines using adjuvanted or nonadjuvanted H1N1 vaccines [19,20]. Zhu et al.  showed that in the elderly individual (>61 years), a second dose of a nonadjuvanted H1N1 vaccine 21 days after the first dose was able to increase the rates of patients with an HAI Ab titer of at least 1: 40 from 79 to 93% (with 15 μg of hemagglutinin) and from 84 to 96% (with 30 μg), respectively.
Most studies have shown lower Ab responses to seasonal influenza vaccination in HIV-infected patients compared with uninfected individuals [6–13]. Lower CD4 cell counts or previous AIDS was associated with limited Ab responses to immunization in some studies [10,12,22,23], though other studies could not identify risk factors for nonresponse in HIV-infected individuals [7,8,13]. Similar to our study, most of these investigations are limited by a low number of patients; especially patients with low CD4+ cell counts were not well represented. In our study, only eight out of 160 patients had a current CD4 cell count less than 200/μl, thus limiting our results to patients with moderate immunodeficiency. Despite a high mean CD4 cell count of all patients included in our study (514 ± 246 cells/μl), higher CD4 cell count was associated with seroconversion (P = 0.03), whereas previous AIDS or a low CD4 nadir was not. Two more recent studies showed vaccine response of the seasonal influenza vaccination to be related to HIV RNA PCR levels [24,25]. The proportion of patients treated with HAART or having a HIV RNA PCR below the limit of detection was higher in the group of seroconverters, but these differences were not statistically significant. Three studies [8,14,15] compared the efficacy of adjuvanted vs. nonadjuvanted influenza vaccines in HIV-infected individuals. In all three studies, the adjuvanted vaccine formulation resulted in a similar or superior immunoresponse; therefore, it seems unlikely that vaccine formulation used in our study explains the low rate of seroconversion.
Patients who had received two doses of nonadjuvanted influenza A/H5N1 vaccination in a trial conducted early in 2009 (ClinicalTrials.gov Identifier: NCT00711295) had a higher rate of H1N1 seroconversion, though Ab titers before H1N1 vaccination were not different. One study examined anti-N1 Abs in patients vaccinated with a contemporary human seasonal influenza H1N1 and found no cross-reactivity to H5N1 . Thus, protective immunity by cross-reactive Ab between H5N1 and H1N1 seems unlikely. One reason for the higher response rate might be an immunogenicity-training effect caused by the previous H5N1 vaccination with an antigen patients had most likely never been exposed to. Whereas seasonal influenza vaccination always contains an antigen-mix that patients may have been exposed to before and therefore does not seem to influence the results. Because of the low number, this finding should be interpreted very cautiously.
It is assumed that virus-specific Abs neutralize the influenza virus by interaction with the viral hemagglutinin. Therefore, the presence of influenza virus-neutralizing Abs serves as a surrogate for immunity to influenza. Favored and standard laboratory method to assess such Abs is the HAI test. It could be that in our assay, detection of specific Ab was less sensitive compared to previous studies and, therefore, would lead to an underestimation of the rate of seroconverters. But as every assay included a standard dilution of antigen in order to titer the value, this would equally affect the results. Furthermore, the percentage of patients with a positive Ab titer as well as the geometric mean titer before vaccination in our study is well in agreement with findings of other studies.
Clinical protection from H1N1, however, rather than Ab response, would be the optimal outcome for a vaccine study. Such studies will be likely to give the best results, but also will take far more time. As the influenza season is still ongoing in the Northern Hemisphere, fast results are required in order to optimally protect HIV-infected patients. Therefore, it needs to be investigated whether a second dose of the available H1N1 vaccine will increase response rates in the patients who did not develop protective H1N1 Ab titers.
R.A. and H.W.D. established and measured the HAI. E.H. and M.B. did the statistical analyses. M.B., N.v.H., C.S. and H.R.B. wrote the protocol. All other authors, including the latter, consented, treated and vaccinated the patients. This study was funded by an unrestricted institutional research grant. There are no conflicts of interest to declare for all the authors.
1. CDC. Use of influenza A (H1N1) 2009 monovalent vaccine: recommendations of the Advisory Committee on Immunization Practices (ACIP), 2009
2. Robert Koch Institut (RKI). Notice of the Permanent Vaccination Committee (Ständigen Impfkommission (STIKO)) of the Robert Koch-Institute for the vaccination against the novel influenza A (H1N1)
. Epid Bull
3. Ada GL, Jones PD. The immune response to influenza virus infection. Curr Top Microbiol Immunol 1986; 128:1–54.
4. Couch RB, Kasel JA. Immunity to influenza in man. Annu Rev Microbiol 1983; 37:529–549.
5. Benne CA, Kroon FP, Harmsen M, Tavares L, Kraaijeveld CA, De Jong JC. Comparison of neutralizing and haemagglutination-inhibiting antibody responses to influenza A virus vaccination of human immunodeficiency virus-infected individuals. Clin Diagn Lab Immunol 1998; 5:114–117.
6. Staprans SI, Hamilton BL, Follansbee SE, Elbeik T, Barbosa P, Grant RM, et al
. Activation of virus replication after vaccination of HIV-1-infected individuals. J Exp Med 1995; 182:1727–1737.
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(suppl 5):B29–B32.
8. Iorio AM, Francisci D, Camilloni B, Stagni G, De Martino M, Toneatto D, et al
. Antibody responses and HIV-1 viral load in HIV-1-seropositive subjects immunised with either the MF59-adjuvanted influenza vaccine or a conventional nonadjuvanted subunit vaccine during highly active antiretroviral therapy. Vaccine 2003; 21:3629–3637.
9. Ragni MV, Ruben FL, Winkelstein A, Spero JA, Bontempo FA, Lewis JH. Antibody responses to immunization of patients with hemophilia with and without evidence of human immunodeficiency virus (human T-lymphotropic virus type III) infection. J Lab Clin Med 1987; 109:545–549.
10. Nelson KE, Clements ML, Miotti P, Cohn S, Polk BF. The influence of human immunodeficiency virus (HIV) infection on antibody responses to influenza vaccines. Ann Intern Med 1988; 109:383–388.
11. Miotti PG, Nelson KE, Dallabetta GA, Farzadegan H, Margolick J, Clements ML. The influence of HIV infection on antibody responses to a two-dose regimen of influenza vaccine. JAMA 1989; 262:779–783.
12. Kroon FP, Rimmelzwaan GF, Roos MT, Osterhaus AD, Hamann D, Miedema F, et al
. Restored humoral immune response to influenza vaccination in HIV-infected adults treated with highly active antiretroviral therapy. AIDS 1998; 12:F217–F223.
13. Brydak LB, Hryniewicz HJ, Machala M, Horban A. Humoral response to influenza vaccination in HIV-infected patients. Clin Drug Invest 1999; 17:441–449.
14. Durando P, Fenoglio D, Boschini A, Ansaldi F, Icardi G, Sticchi L, et al
. Safety and immunogenicity of two influenza virus subunit vaccines, with or without MF59 adjuvant, administered to human immunodeficiency virus type 1-seropositive and-seronegative adults. Clin Vaccin Immunol 2008; 15:253–259.
15. Gabutti G, Guido M, Durando P, De Donno A, Quattrochi M, Bacilieri S, et al
. Safety and immunogenicity of conventional subunit and MF59-adjuvanted influenza vaccines in human immunodeficiency virus-1-seropositive patients. J Int Med Res 2005; 33:406–416.
17. European Committee for Proprietary Medicinal Products. Note for guidance on harmonisation of requirements for Influenza vaccines (CPMP/BWP/214/96).
London: European Agency for the Evaluation of Medicinal Products; 1997.
18. Center for Biologics Evaluation and Research. Guidance for industry: clinical data needed to support the licensure of pandemic influenza vaccines. Bethesda, MD: Food and Drug Administration; May 2007.
19. Gross PA, Weksler ME, Quinnan GV Jr, Douglas RG Jr, Gaerlan PF, Denning CR. Immunization of elderly people with two doses of influenza vaccine. J Clin Microbiol 1987; 25:1763–1765.
20. Levine M, Beattie BL, McLean DM. Comparison of one- and two-dose regimens of influenza vaccine for elderly men. CMAJ 1987; 137:722–726.
21. Zhu FC, Wang H, Fang HH, Yang JG, Lin XJ, Liang XF, et al
. A novel influenza A (H1N1) vaccine in various age groups. N Engl J Med 2009; 361:2414–2423. Epub 2009 Oct 21.
22. Fowke KR, D'Amico R, Chernoff DN, Pottage JC Jr, Benson CA, Sha BE, et al
. Immunologic and virologic evaluation after influenza vaccination of HIV-1-infected patients. AIDS 1997; 11:1013–1021.
23. Iorio AM, Alatri A, Francisci D, Preziosi R, Neri M, Donatelli I, et al
. Immunogenicity of influenza vaccine (1993–94 winter season) in HIV-seropositive and -seronegative ex-intravenous drug users. Vaccine 1997; 15:97–102.
24. Evison J, Farese S, Seitz M, Uehlinger D, Furrer H, Muhlemann K. Randomized, double-blind comparative trial of subunit and virosomal influenza vaccines for immunocompromised patients. Clin Infect Dis 2009; 48:1402–1412.
25. Yamanaka H, Teruya K, Tanaka M, Kikuchi Y, Takahashi T, Kimura S. Efficacy and immunologic responses to influenza vaccine in HIV-1-infected patients. J Acquir Immune Defic Syndr 2005; 39:167–173.
26. Frobert E, Bouscambert-Duchamp M, Escuret V, Mundweiler S, Barthélémy M, Morfin F, et al
. Anti N1 Cross-protecting antibodies against H5N1 detected in H1N1 infected people
. Curr Microbiol
2009 [Epub ahead of print].
This article has been cited 7 time(s).
Human Vaccines & ImmunotherapeuticsImmune response to 2009 H1N1 vaccine in HIV-infected adults in Northern ThailandHuman Vaccines & Immunotherapeutics
Medical Microbiology and Immunology"Trivalent influenza vaccination of healthy adults 3 years after the onset of swine-origin H1N1 pandemic: restricted immunogenicity of the new A/H1N1v constituent?"Medical Microbiology and Immunology
VaccineRelation of activation-induced deaminase (AID) expression with antibody response to A(H1N1)pdm09 vaccination in HIV-1 infected patientsVaccine
VaccineHIV-infected patients show impaired cellular immune response to influenza vaccination compared to healthy subjectsVaccine
HIV Clinical TrialsImmune Response after a Single Dose of the 2010/11 Trivalent, Seasonal Influenza Vaccine in HIV-1-Infected Patients and Healthy ControlsHIV Clinical Trials
AIDS Research and Human RetrovirusesCellular and Humoral Immune Responses to Pandemic Influenza Vaccine in Healthy and in Highly Active Antiretroviral Therapy-Treated HIV PatientsAIDS Research and Human Retroviruses
Clinical Infectious DiseasesImmunogenicity and Safety of the Human Papillomavirus 6, 11, 16, 18 Vaccine in HIV-Infected Young WomenClinical Infectious Diseases
H1N1; hemagglutination inhibition assay; HIV; immunoresponse
© 2010 Lippincott Williams & Wilkins, Inc.
What does "Remember me" mean?
By checking this box, you'll stay logged in until you logout. You'll get easier access to your articles, collections,
media, and all your other content, even if you close your browser or shut down your
To protect your most sensitive data and activities (like changing your password),
we'll ask you to re-enter your password when you access these services.
What if I'm on a computer that I share with others?
If you're using a public computer or you share this computer with others, we recommend
that you uncheck the "Remember me" box.
Highlight selected keywords in the article text.
Data is temporarily unavailable. Please try again soon.