In HIV-infected patients, vaccination against flu is recommended annually due to the higher risk for complications related to infection . However, numerous studies demonstrated a lower immunogenicity of both seasonal influenza and pandemic 2009 A/H1N1v vaccines in HIV-1-infected patients [2,3]. Because the influenza season varies in timing and duration, durability of seroprotection is also an important parameter for influenza vaccines . Data on durability of the immune response conferred by seasonal or pandemic influenza vaccines administered to HIV-1-infected patients are, however, lacking. A recent article reported that a single dose of a nonadjuvanted monovalent 2009 influenza A did not generate durable antibody response in HIV-1-infected patients . Thus, in HIV-1-infected patients, alternatives schedules more immunogenic than the standard flu vaccine regimen are needed to enhance and prolong vaccine efficacy.
We reported recently in HIV-1-infected adults that a two-dose schedule of 2009 A/H1N1v vaccine was more immunogenic than a single dose 1 month after the vaccination; moreover, the AS03A-adjuvanted H1N1v vaccine yielded a higher short-term immune response than the nonadjuvanted vaccine with no impact on HIV infection . We now report the durability of the immune response up to 1 year after vaccination.
The HIFLUVAC trial was a phase 2, randomized, patient-blinded trial conducted in 18 ANRS-labeled centers in France as described previously .
Participants were HIV-1-infected adult patients, either receiving HAART for at least 6 months (HIV viral load <50 copies/ml) or without antiretroviral therapy for at least 6 months. Patients were eligible to participate if they did not have an history of influenza infection during the previous 6 months, had no febrile episode within 1 week prior to vaccination, did not receive immune-modulatory or immunosuppressant drugs, had no progressive opportunistic infection during the previous month and no other immunization within 3 weeks prior to inclusion.
Written informed consent was obtained from each patient. The protocol was conducted in accordance with the Declaration of Helsinki and French law for biomedical research and was approved by the local Ethics Committee (‘Comité de Protection des Personnes Ile-de-France III’, Paris).
Patients were centrally randomized (1 : 1) to receive two intramuscular injections of 2009 A/H1N1v vaccine formulated with (group A) or without AS03A-adjuvant (group B), administered 21 days apart. Patients were stratified according to HAART vs. no HAART at baseline.
Blood samples were planned for assessment of immunogenicity at day 0 prior to vaccination, 21 days following each dose, and at months 6 and 12. Standard biochemical tests, CD4/CD8 cell counts and serum HIV-1 RNA were planned at days 0, 21, 42 and months 3, 6, 12.
Monovalent A/H1N1v inactivated, split-virion vaccine (FLU D-PAN H1N1 [A/California/7/2009 (H1N1)v]) was manufactured by GlaxoSmithKline (GSK) Biologicals (Dresden, Germany). AS03A-adjuvanted H1N1v was formulated with 3.75 μg haemagglutinin and AS03A, a tocopherol oil in-water emulsion adjuvant system manufactured by GSK Biologicals; nonadjuvanted vaccine was formulated with 15 μg haemagglutinin. The 2009 A/H1N1v vaccine was injected intramuscularly, with 0.5 ml of antigen suspension mixed or not with AS03A adjuvant emulsion.
Serum haemagglutination inhibition antibodies have been analyzed in a centralized laboratory (GSK Biologicals). A validated microtiter haemagglutination inhibition test was used with chicken erythrocytes and with the A/California/7/2009 (H1N1)v strain as antigen . The sample titer was the highest dilution that completely inhibited haemagglutination.
Virus neutralization by antibodies contained in the serum was determined in a micromethod assay (Viroclinics BV, Rotterdam, The Netherlands) . Sera were tested for the presence in serum of neutralizing antibodies against influenza viruses A/Netherlands/602/2009 pandemic H1N1 influenza virus (a A/California/07/2009-like virus).
All laboratory assays were performed by personnel blinded to vaccine allocation.
According to the regulatory authorities in Europe and the USA [9,10], endpoints for haemagglutination inhibition antibody response in adults 18–60 years are seroprotection rate (postvaccination haemagglutination inhibition titer ≥1 : 40); seroconversion rate (prevaccination haemagglutination inhibition titer <1 : 10 and postvaccination titer ≥1 : 40, or prevaccination titer ≥1 : 10 and at least a four-fold increase in postvaccination titer); seroconversion factor as the geometric mean of the within-patient ratios of the postvaccination reciprocal haemagglutination inhibition titer to the Day 0 reciprocal haemagglutination inhibition titer.
For the neutralizing antibodies, seroconversion was defined as prevaccination titer less than 1 : 8 and postvaccination titer at least 1 : 32, or prevaccination titer at least 1 : 8 and at least a four-fold increase in postvaccination titer.
As planned, analyses were descriptive and presented vaccinated and tested patients for immunogenicity. The 95% confidence intervals (CI) for rates were exact (Clopper–Pearson) CIs. Logistic regressions were used to determine the factors associated with haemagglutination inhibition titers at least 1 : 40.
Among the 309 patients randomized between October 26 and November 6 2009, 306 received a first dose of vaccine and 296 received the two doses. The long-term immunogenicity was assessed in 91% of patients at months 6 and 12, for whom two doses were received and haemagglutination inhibition assay results were available.
The demographic profiles and the clinical characteristics of the two randomized groups were well balanced as described previously . Median age was 47 years, 19% of patients were women and 23% were classified as Centers for Disease Control and Prevention stage C. Patients receiving HAART were 77% and median CD4 cell counts were above 500 cells/μl for both HAART-treated and untreated patients. Thirty-one percent of patients were found to have received the 2009 seasonal influenza vaccine, at least 21 days before 2009 A/H1N1v first vaccine injection. Only 20 patients (7%) have received the 2010 seasonal influenza vaccine containing the 2009 A/H1N1v strain, 10 days in median before month 12 (range: 1–55 days).
From the first vaccine injection until month 12, serious adverse events were reported in 13% of patients in each randomized group and none was related to H1N1v vaccination. No significant change in HIV viral load was observed and CD4 cell count levels remained stable throughout the follow-up in both groups (data not shown). In patients receiving HAART, low levels of HIV-1 RNA were observed at 3 weeks after vaccination in four (3%) and eight (7%) patients in groups A and B, respectively.
The rates of patients with haemagglutination inhibition antibody titers of 1 : 40 or greater were 7.7 and 10.0% at day 0, 98.6 and 92.1% at day 42, 83.7 and 59.4% at month 6, 70.4 and 49.3% at month 12, in group A and group B, respectively. The geometric mean titers (GMT) for haemagglutination inhibition antibodies were 8.5 and 8.1 at day 0, 381.9 and 171.1 at day 42, 82.0 and 46.7 at month 6, 63.4 and 39.5 at month 12, respectively (Table 1, Fig. 1).
In a univariate and multivariate analysis, the predictors of persistence of haemagglutination inhibition titers of 1 : 40 or greater at months 6 and 12 were the AS03A-adjuvanted H1N1v vaccine [odds ratio (OR) = 3.8, P = 0.00001 at month 6; OR = 2.7, P = 0.0002 at month 12), HAART (OR = 2.0, P = 0.028 at month 6; OR = 1.7, P = 0.15 at month 12) and current smoking (OR = 0.55, P = 0.04 at month 6; OR = 0.57, P = 0.03 at month 12) (details are presented under Supplemental Digital Content, http://links.lww.com/QAD/A259). Seasonal 2010 influenza vaccination was independently associated with haemagglutination inhibition titers of 1 : 40 or greater at day 364 (OR = 5.6, P = 0.026).
Microneutralization results were overall comparable to haemagglutination inhibition results. However, the microneutralization antibody titers were slightly lower than haemagglutination inhibition titers as well as seroconversion rates (Table 1, Fig. 1).
In this randomized trial designed to evaluate the immunogenicity and the safety of 2009 A/H1N1v vaccine, adjuvanted or not with AS03A, in a population of HIV-1-infected adults, we observed that two doses of the adjuvanted vaccine formulated with 3.75 μg of haemagglutinin antigen confers a higher long-term immune response than two doses of the nonadjuvanted vaccine.
The results of Crum-Cianflone et al. showed that a single dose of monovalent 2009 influenza A/H1N1v nonadjuvanted vaccine did not generate durable seroprotective antibody response in HIV-1-infected patients. Only 43% of them had a postvaccination titer at least 1 : 40 at month 6 vs. 73% in healthy adults. In the general population, with a trivalent seasonal vaccine including one H1N1 strain, seroprotection rates were maintained above European criteria at 6 months and decreased to 42–52% after 1 year in adults less than 65 years . Our results suggest that a second dose of nonadjuvanted vaccine in HIV-1-infected patients could improve long-term seroprotection up to 59.4% at month 6. One year after vaccination, 43.3% of them had seroprotective levels. This suggests that vaccination with two doses of nonadjuvanted H1N1v vaccine in HIV-1-patients with controlled virologic parameters allows achieving comparable long-term seroprotection rates as one dose in the general population.
By comparison, with two doses of adjuvanted vaccine, seroprotection levels as high as 83.7% (89.9% in patients with HAART) were achieved at month 6. However, recent results showed higher seroprotection rate as high as 96.6% in healthy adults receiving two doses of adjuvanted vaccine . One year after vaccination, seroprotection rates in our study remained high at 70.4% (75.0% in patients with HAART). Better seroprotection rates were obtained in patients with HAART as reported previously in case of seasonal influenza vaccination . Our study demonstrates that this benefit is observed up to 1 year after 2009 A/H1N1v vaccination.
Neutralizing antibody responses were also observed in both groups after vaccination. Although the overall kinetics of the neutralizing antibody response paralleled that of the haemagglutination inhibition response, lower GMTs and seroconversion rates were observed 6 and 12 months postvaccination. Such variation may partly correspond to different biological activities of the antibodies measured by the two methodologies. However, neutralizing seroconversion rates observed in healthy adults receiving two doses of adjuvanted vaccine were as high as 76% at month 6 as compared to 43.2% (54.3% in patients with HAART) in our study .
Influenza vaccination has been shown to cause transient increases in plasma HIV-1 RNA levels. In a Swiss study, this was observed in previously aviraemic patients after two doses of the AS03A-adjuvanted H1N1v vaccine . A subsequent nonadjuvanted dose of a trivalent split-virus influenza vaccine, containing the 2009/A/H1N1v strain did not reproduce this finding when administered 1 year after AS03A-adjuvanted H1N1v vaccination, and, therefore, the authors concluded on a nonantigen-specific adjuvant effect. Although our study was not designed to precisely evaluate this outcome, no significant impact on HIV-1 RNA or CD4 levels were found, whatever the vaccine used.
Our study has some limitations. The present results were based on accepted immunological surrogates but other components of the immune response have not yet been explored. T-cell-mediated immune response is known to play a critical role in host defense against influenza virus infection. Therefore, assuming protection only on the basis of humoral response could be questioned, particularly in immunosuppressed patients. The results of a recent seasonal influenza vaccination trial done in Africa in HIV-1-infected patients suggested that the likely benefit of influenza immunization was underestimated when relying only on humoral immunogenicity markers .
In conclusion, our long-term results in HIV-1-infected patients demonstrate that two doses of nonadjuvanted vaccine in HIV patients achieve long-term seroprotection rates comparable to the general population after a single dose. Administration of two doses of adjuvanted influenza vaccine maintains higher seroprotection titers up to 1 year after vaccination than two doses of nonadjuvanted vaccine, however, lower than in the general population.
We thank the study participants and the participating clinicians at each site, the ‘French Research Program on Pandemic H1N1v Flu: Institut de Microbiologie et Maladies Infectieuses’, Francis Beauvais for his help in preparing the manuscript; GSK Biologicals staff (Jeanne-Marie Devaster, F.R., Laurence Baufays, Nathalie Clyti, Sophie Muller, Marie-Hélène Chautard, and Isabelle Naeije for support in study management, Karl Walravens, Urban Lundberg, and Roger Bernhard for serological testing, and Géraldine Drevon for manuscript review). Trial registration: Clinicaltrials.gov identifier: NCT01008813.
O.L. and J.P.A., C.Desaint and C.Durier prepared the first draft of the manuscript. All authors contributed to the content of the manuscript (including the author employed by GSK Biologicals) and to the design and conduct of the study; the analysis and interpretation of the data; and the preparation of the manuscript. C. Durier performed statistical analysis. O.L. had final responsibility for the decision to submit the manuscript for publication.
ANRS 151 HIFLUVAC study group members: G. Pialoux, L. Slama (Hôpital Tenon, Paris), J.F.D., J. Ghosn, M.T. Rannou, E. Fourn, Y. Quertainmont (Hôpital de Bicêtre, Le Kremlin-Bicêtre), F. Raffi, C. Allavena, C. Biron, D. Besson, H. Hue (Hôpital de l’Hôtel-Dieu, Nantes), J. Reynes, J.M. Jacquet (Hôpital Gui de Chauliac, Montpellier), J.M. Molina, N. Colin de Verdière (Hôpital Saint-Louis, Paris), P.M.G., M.C. Meyohas, D. Bollens (Hôpital Saint-Antoine, Paris), P. Yéni, X. Duval, B. Phung, C. Godard, N. El Alami albi (Hôpital Bichat-Claude Bernard, Paris), O.L., P. Loulergue, L. Belarbi, L. Iordache (Hôpital Cochin, Paris), A. Sobel, Y.L., E. Bamago, S. Doninguez, C. Dumont, C. Chesnel (Hôpital Henri Mondor, Créteil), H. Laurichesse, J. Beytout, C. Jacomet, (Hôpital Gabriel Montpied, Clermont Ferrand), D. Rey, J.M. Lang, P. Fischer, M. Partisani (Hôpitaux Universitaires, Strasbourg), Y. Yazdanpanah, F. Ajana, T. Huleux (Hôpital Gustave Dron, Tourcoing), F.L., C. Delafontaine, C. Guglielminotti, A. Frésard, P. Fouilloux, V. Ronat (Hôpital Bellevue, Saint-Etienne), T.M., S. Wassoumbou (Hôpital de Brabois, Vandoeuvre-lès-Nancy), J-F. Bergmann, A.R., M. Parrinello (Hôpital Lariboisière, Paris), C.M., F. Fily, M. Ratajczak (Hôpital Pontchaillou, Rennes), F. Bricaire, C. Katlama, A. Chermak (Hôpital Pitié-Salpêtrière, Paris), P. Dellamonica, A. Leplatois (Hôpital de l’Archet, Nice), J. Saillard, S. Couffin-Cadiergues, A. Bouxin-Métro (ANRS, Paris), J.P.A., C. Desaint, V. Foubert, M. Resch, A. Grenier, M. Le Cornec, E. Moreau, S. Marie-Antoine, Y. Saïdi, A. Arulananthan, C. Durier (Methodology and coordinating center, INSERM SC10, Villejuif France).
REIVAC Network: O.L. (coordinator: CIC BT505, Paris), F.L. (CIC CIE3 axe vaccinologie, Saint Etienne), X. Duval (CIC P-007, Bichat-Claude Bernard, Paris), N. Colin de Verdière (Service de maladies infectieueses et tropicales de Saint-Louis, Paris), H. Laurichesse, J. Beytout (CIC P-501, Clermont Ferrand).
Scientific committee: O.L., coordinator (Université Paris Descartes, Hôpital Cochin, Inserm, Paris); J.P.A., C. Durier, C. Desaint (Inserm SC10, Villejuif); Pierre Loulergue (Hôpital Cochin, Paris); Xavier Duval (Hôpital Bichat, Paris); Christine Jacomet (CHU Clermont Ferrand, Clermont Ferrand); Constance Delaugerre (Hôpital Saint-Louis, Paris); Sylvie van der Werf (Institut Pasteur, Paris); Sophie.Muller (GSK, Paris); F.L. (Hôpital Bellevue, Saint-Etienne), Jade Gohsn, J.F.D. (Hôpital de Bicêtre, Kremlin-Bicêtre), Marianne Lhenaff (TRT5, Paris), Juliette Saillard, Sandrine Couffin-Cadiergues (ANRS, Paris).
This study was sponsored and funded by the French National Agency for Research on AIDS and Viral Hepatitis (ANRS, Paris, France). The sponsor of this study did not impose any impediment on the publication of the study's results.
GSK Biologicals provided the vaccines and performed immunological tests.
Conflicts of interest
O.L. has been an investigator on vaccine studies sponsored by GlaxoSmithKline Biologicals and other companies and received travel support to attend scientific meetings from GlaxoSmithKline and other companies. F.R. is an employee of GSK Biologicals and has shares from GSK Biologicals. C.D., C.D., F.L., P.M.G., Y.L., T.M., C.M., A.R., C.K., A.G., J.F.D., J.P.A. have no conflicts of interest.
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