In April 2009, a new H1N1 influenza A virus was isolated from people in Mexico and the USA [1,2]. The new strain was characterized by highly efficient human-to-human transmission to justify WHO to declare the emergence of a new influenza pandemic on 11 June 2009 . The molecular characterization of this new H1N1 virus highlighted the unique combination of gene segments from both North America and Eurasian influenza virus of swine origin (S-OIV) . The antigenic distinction of S-OIV from seasonal influenza A viruses raised the concern that the human population lacked protective immune memory. Indeed, neutralizing antibodies (Nabs) against S-OIV were found nearly exclusively in persons born before 1957 .
To limit the spread of S-OIV, a prompt response was engaged by developing a novel monovalent vaccine against the virus strain A/California/07/2009 (H1N1). As a large proportion of the human population does not possess cross-NAb, two doses of the vaccine, according to the so-called ‘Prime-Boost’ protocol, were planned. Preliminary small studies, however, showed that one dose was necessary and sufficient to obtain antibody titers considered protective in healthy adults, that is haemagglutination inhibition (HAI) titers higher than 40 [5,6]. Further large studies confirmed that a single vaccine dose was highly immunogenic in healthy adults, as more than 90% of vaccinees seroconverted and developed ‘protective’ antibody titers [7–10].
Despite these excellent results, the safety and immunogenicity of the new monovalent vaccine has not been reported in immunosuppressed hosts. A recent report has shown that HIV-1-infected individuals vaccinated with adjuvanted pandemic H1N1 vaccine show low rates of seroconversion, although a control group of healthy seronegative individuals was not included in this study . This study is consistent with a number of previous trials on seasonal influenza vaccination showing that postvaccination antibody titers were significantly lower in HIV-1-positive individuals vs. uninfected controls [12,13]. Indeed, the weak antibody response to seasonal influenza vaccination of HIV-1-positive individuals was correlated to a poor B-cell memory response rather than a plasma cell response, suggesting that the HIV-1-positive individuals may initially respond to immunization, but that further differentiation or survival of memory B cells was impaired during disease progression . Although the antibody titers of HIV-1-positive individuals did not reach the values of those of uninfected individuals, their vaccination with adjuvanted seasonal vaccine preparation was reported to increase the immunogenicity as compared with nonadjuvanted vaccines [14,15]. As influenza has a significant morbidity among HIV-1-positive individuals, including those treated with HAART , international guidelines recommend that HIV-1-positive individuals should be vaccinated against influenza annually. In consideration of the risk that HIV infection might increase the risk of serious illness from 2009 H1N1 influenza , the pandemic A/H1N1 2009 vaccine was administered to HIV-1-positive individuals in a single dose following the same recommendations applied to healthy individuals.
In the absence of immunogenicity studies in HIV-1-positive individuals, we investigated whether a single dose of this monovalent MF59-adjuvanted H1N1 vaccine could induce an effective antibody response in HIV-1-positive individuals, whether the immune response would be similar to that of HIV-negative individuals and, finally, whether predictive factors of seroconversion and seroprotection could be identified.
Participants and methods
Participants and procedures
HIV-infected volunteers of both sexes were recruited between mid-November and December 2009 at the Infectious Diseases Clinic of San Raffaele Scientific Institute (HSR) whereas the HIV-1-negative healthy volunteers were enrolled among healthcare workers (HCW) of both HSR and Desio Hospital (Milan, Italy). The eligibility criteria were identical for the HIV-1-positive and HIV-1-negative participants, except for the HIV status. We excluded participants below 18 and above 65 years of age, pregnant women and individuals who displayed any known or suspected allergy to a vaccine component. The Institutional Review Boards of HSR and Desio Hospital approved all procedures. Following written informed consent signature, individuals entering the study were interviewed. Detailed clinical and prior vaccination histories were collected. All participants donated a blood sample (T0) prior to receiving the pandemic influenza MF59-adjuvanted H1N1v vaccine. A single intramuscular dose injection in the deltoid of licensed monovalent vaccine (Focetria; Novartis Vaccines, Siena, Italy), formulated to contain 7.5 μg of haemagglutinin antigen of the strain A/California/7/2009 (H1N1)v like strain (X179A), oil-in-water emulsion of MF59C.1 adjuvant was administered to all participants. Each individual was monitored after vaccination for adverse events occurring within 1 h after vaccination. Volunteers donated a second blood sample 3–5 weeks postvaccination (T28).
Immunogenicity of the adjuvanted vaccine
Serum was separated from whole venous blood collected from the individual before (T0) and 3–5 weeks (T28) after vaccination and stored at −80°C until use. Antibody against the S-OIV antigens were measured by the standard HAI assay on stored sera . Briefly, serum samples were treated with receptor-destroying enzyme (RDE; Denka Seiken Co. Ltd, Tokyo, Japan) at 37°C for 16 h. Two-fold serial dilution were mixed with 0.5% turkey erythrocytes (Charles River, Calco, Italy) for 1 h at room temperature in 96-well plates. The lowest dilution of serum that could be tested was 1: 4. All samples were tested in blind and in duplicate. The HAI titer was defined as the reciprocal of the highest serum dilution that completely inhibited agglutination. Titers higher than 40 were considered ‘protective’; therefore, the seroprotection rate was defined as the proportion of individuals who had titers higher than 40, whereas the seroconversion rate was the proportion of participants with HAI prevaccination titer less than 1: 10 and postvaccination titer more than 1: 40 or a prevaccination titer more than 10 and an increase of at least four-fold or more after vaccination .
No sample size calculation was performed a priori and individuals who decided to undertake vaccination against pandemic A/H1N1v influenza between mid-November and December 2009 and voluntarily donated a blood sample prior and postvaccination were included in this study. Titer values were used for the calculation of geometric mean titers (GMTs) and 95% confidence interval (CI). The reverse cumulative distribution function (RCDF) was calculated on log10-transformed titers for HIV-1-positive patients and HIV-1-negative individuals before and after vaccination. To analyze whether the HIV-1 infection status or clinical and demographic characteristics might have an effect on GMTs before and after vaccination, either a χ2-test or a Wilcoxon rank-sum test were used when dealing with categorical or continuous variables, respectively.
Logistic regression was also applied to assess the influence of clinical and demographic characteristics on both seroprotection and seroconversion rates. When the outcome was the seroprotection rate, only a univariable analysis was performed as the majority of both HIV-negative and HIV-positive individuals were seroprotected (> 97%) after vaccination, whereas both univariable and multivariable logistic regressions were calculated for the seroconversion rate. Odds ratio (OD) as well as the corresponding 95% CI were estimated for each covariate considered in the study. In regard to the multivariable analysis, only the characteristics available for both in HIV-1-positive and HIV-1-negative individuals were included into the model (entry criteria: univariable P-value ≤ 0.05). All reported P-values were two-sided; analyses were performed using the SAS software version 9.2 (SAS Institute, Cary, North Carolina, USA).
Demographic features of the study population and characteristics of HIV-1-positive individuals
Table 1 shows the demographic characteristics of the two study populations, that is HIV-1-positive and HIV-1-negative. The age of the HIV-1-positive was higher than that of the HIV-1-negative individuals (mean: 45.2 vs. 39.4 years old), whereas no significant difference was detected in relation to sex between the two groups.
The baseline characteristics of the HIV-1-positive individuals are listed in Table 1. All participants were on HAART with 34 of 44 patients showing less than 50 copies of plasma HIV-RNA/ml with the remainders having log10 viremia levels of 3.05, 95% CI 2.16–3.19 log10 copies/ml. The baseline mean viremia of all 44 HIV-1-positive individuals was 1.97 log10 copies/ml 95% CI 1.75–2.19.
From prior vaccination histories, a higher proportion of HIV-1-positive individuals received the 2009 seasonal vaccine as compared with the HIV-1-negative individuals (27.9 vs. 7.4%, respectively), whereas the latter group tended to have a higher frequency of seasonal vaccination in years prior to 2009 than the HIV-1-positive group (65 vs. 47.7%, respectively) consistently with being HCW. The day of postvaccination blood sample collection was planned to occur within 3–5 weeks after immunization, the mean day ± SD of blood sampling are indicated in Table 1. For simplicity, T28 was used for both the HIV-1-positive and HIV-1-negative groups throughout.
Safety and immunogenicity of the adjuvated vaccine
Neither serious adverse events nor dropouts related to adverse events occurred during the study in both HIV-1-positive and HIV-1-negative groups. Furthermore, no significant increase in HIV viremia was observed after immunization when data obtained at T28 postvaccination were compared with those of baseline (Table 1).
At baseline, the GMT values of antibody against the vaccine strain were significantly higher in HIV-1-positive than in HIV-1-negative individuals (73.7 vs. 41.2, respectively, P < 0.0001) (Table 2). A higher seroprotection rate in HIV-1-positive as compared with the HIV-1-negative individuals was observed (79.6 vs. 35.1%, respectively, P < 0.0001; Table 1 and Fig. 1, left panel). The analysis of postvaccination antibody titers showed that more than 97% of the individuals reached seroprotection antibody titers regardless of the HIV infection status (Table 2). Although the T28 GMT values were apparently higher in HIV-1-negative than in HIV-1-positive individuals, no statistically significant differences were observed and the RCDF curves of the postvaccination antibody titers of the two groups were superimposable (Fig. 1, right panel). The proportion of individuals who seroconverted was higher in the HIV-1-negative than in the HIV-1-positive group (Table 2).
Next, we analyzed whether additional factors, other than the HIV-1 status, could have influenced the achievement of seroprotection antibody levels after vaccination (Table 3). Among the demographic features of the participants, older ages significantly reduced (OR = 0.595, 95% CI 0.330–0.942, P = 0.049) the likelihood of possessing seroprotection titers (> 40), whereas none of the factors related to HIV infection, such as CD4+ nadir, CD4+ T-cell numbers, CD4+ and CD8+ ratio, HIV-RNA load and total lymphocytes, influenced this parameter. Prior seasonal influenza vaccination was not associated with seroprotection after vaccination, whereas increasing baseline levels of HAI titers were associated with a higher probability of achieving seroprotection postvaccination (OR = 500.2, 95% CI 19.9 to > 999.9, P = 0.002).
Finally, multivariable logistic regression was applied to estimate the independent contribution of the demographic and immunization characteristics that resulted to be predictive of the seroconversion condition in the univariable analysis. As shown in Table 4, both age and lack of HIV-1 infection significantly influenced the likelihood of being seroconverters. Indeed, individuals with increasing age (OR = 0.805, 95% CI 0.684–0.947, P = 0.009) or HIV-1-positive condition (OR = 0.237, 95% CI 0.104–0.539, P = 0.0006) were less likely able to seroconvert after influenza vaccination.
In the present study, we show the MF59-adjuvanted H1N1 vaccine induces antibody titers in both HIV-1-positive individuals and HIV-1-negative individuals. The seroconversion rate, however, was lower in the HIV-1-positive individuals as compared with the HIV-1-negative individuals because the former were characterized by baseline (T0) titers higher than those of the control group. Among other potential factors, only the HIV infection status and an older age (per 5-year increase) were negatively correlated with antiinfluenza antibody seroconversion rates.
The prompt responses to the 2009 pandemic H1N1 influenza had made available a monovalent vaccine in a record time. Unexpectedly, an injection of either adjuvanted or nonadjuvanted monovalent influenza A (H1N1) vaccine was shown to trigger sufficient protection against the pandemic 2009 influenza virus in healthy adults in the range of 18–64 years of age [5,7–10]. As immunocompromised groups have an increased risk of influenza illness and mortality, this vaccination was extended to these groups, including HIV-1-positive individuals, although no clinical trial was reported. Particularly, we focused on HIV-1-positive individuals, as it is well established that their memory B-cell response is impaired in comparison to healthy HIV-1-negative individuals  and their antibody response to seasonal influenza vaccine is less efficient than in nonimmunocompromised hosts [13–15]. In this regard, we have observed that more than 70% of HIV-1-positive individuals possessed antibody titers higher than 40, considered to be seroprotection against the vaccine strain prior to vaccination (Table 2 and Fig. 1, left panel). As our cohort of HIV-1-positive individuals was vaccinated in mid-December 2009 after the peak of 2009 influenza A/H1N1 spreading (http://www.euroflu.org), we cannot exclude that some participants might have been exposed and/or infected with pandemic flu in the previous months. An alternative explanation might be related to the limitations of the standard HAI method to determine antibody titers. We indeed detected higher baseline HAI titers than previously reported . This difference might depend on variations of the HAI methodological procedure concerning the sera treatment with RDE in our study or turkey red blood cells treatment with Alsever's solution in the study by Bickel et al. . Nevertheless, we observed a significantly higher GMT for the HIV-1-positive individuals compared with the HIV-1-negative individuals at baseline. However, the GMT of the postvaccination (T28) antibody titers was similar to those of the HIV-1-negative individuals (215.3 vs. 275.9, respectively, P = 0.123). Likely, the high levels of preexisting antibody has determined the fact that the GMT ratio of T28: T0 was significantly lower in HIV-1-positive vs. HIV-1-negative individuals (2.92 vs. 6.69, respectively, P < 0.0001). Consequently, the proportion of individuals who seroconverted was significantly lower in the HIV-1-positive as compared with the HIV-1-negative group.
Indeed, the multivariable analysis of risk factors pointed out a negative association between seroconversion rate and HIV status and age. We believe that these results do not indicate a poor immunogenicity of the H1N1 2009 vaccine in HIV-1-positive individuals. In contrast, the MF59-adjuvanted H1N1 2009 vaccine induced high immune responses, which meet the European regulatory acceptance criteria . It is possible that the inclusion of the MF59 adjuvant in the vaccine composition might have been responsible for increasing the quantity of specific antibody against the pandemic vaccine strain and, perhaps, the quality of the antibody response by shifting the antibody from antihaemagglutin 2 to antihaemagglutin 1, as recently reported after the immunization against H5N1 influenza virus . The high immune responses observed in HIV-1-positive individuals could also be correlated to the immunological restoration induced by HAART. All HIV-1-positive participants were indeed under HAART, as demonstrated by the high proportion of individuals (34 of 44 individuals) with undetectable viremia levels (< 50 copies of RNA/ml) and no increase in the plasma HIV-1-RNA was observed at T28 postvaccination (Table 1), although we cannot exclude that ‘blips’ of plasma HIV-RNA might have occurred earlier than T28 as a consequence of the immune cell activation induced by the vaccination . Indeed, it has been reported that HAART plays an important role in maintaining or restoring robust B-cell immunological responses to both vaccines and following natural infection [19,22].
Before our study, a recent report has underscored a low rate of seroconversion after vaccination with a single dose of inactivated 2009 H1N1 influenza vaccine with AS03 adjuvant in HIV-1-infected individuals , suggesting that two vaccinations might be needed to achieve comparable seroconversion/seroprotection levels in this population. However, the amount of haemagglutin antigen included in the vaccine preparation used in this study was lower than the one used in our study (3.75 vs. 7.5 μg, respectively). Furthermore, the prevaccination antibody titers of our HIV-positive participants were significantly higher than those reported by Bickel et al. , likely reflecting a higher exposure/infection to pandemic H1N1 influenza virus prior to vaccination in our HIV-positive individuals. In regard to prior vaccination, our participants did not previously receive H5N1 vaccine preparations; however, a fraction of the HIV-positive individuals received the 2009 seasonal vaccine preparation and seasonal vaccines in the previous years, that is 27.9 and 47.7%, respectively (Table 1), which did not interfere with the antibody responses against the 2009 pandemic A/H1N1v influenza vaccine. Previous studies on seasonal vaccination have demonstrated that consecutive vaccinations have a negative impact on the antibody responses [23,24], however, neither seroprotection nor seroconversion rates were influenced by prior vaccination, suggesting that no cross-protective memory antibody responses preexisted in these study individuals prior to the 2009 pandemic vaccine. One last consideration is the age. The HIV-1-positive individuals included in our study were younger than those included in the study by Bickel et al. ; however, increasing age negatively influenced the seroconversion rate in both the HIV-1-positive and HIV-1-negative group consistently with previous reports on seasonal influenza vaccination . Although the eligibility criteria included participants above 18 and below 65 years of age, both HIV-1-negative and HIV-1-positive volunteers were compacted in the fourth and fifth decade of life, respectively. This factor together with the low numbers in each group and the older age of the HIV-1-positive individuals poses caveats and limitations to our study. Worthy of note is the potential bias of recruiting only highly motivated individuals with a more careful health behavior that may likely affect the results in both HIV-1-positive and HIV-1-negative groups.
In conclusion, although HIV infection and increasing ages were associated with a diminished probability of seroconversion, a single dose of monovalent MF59-adjuvanted 2009 influenza H1N1 vaccine containing 7.5 μg of haemagglutin antigen induced an immune response against pandemic H1N1 in HIV-1-positive individuals, reaching titers similar to those of HIV-1-negative individuals.
This work was supported by the Italian Ministry of Health, grant no. RF-FSR-2007–644129 and by Cariplo Foundation, grant no. 2009-3594(to E.V.). The authors wish to thank the volunteers for their participation in this study. We also thank Mrs Anna Biancardi, Paola Nizzero and Liviana Della Torre for assisting the participants during the study. A.M. performed some experiments of the study as partial fulfillment of his pre-MD degree internship at the School of Medicine, Vita-Salute University of Milan, Italy. We acknowledge Professor Guido Poli and Professor Franco Toffoletto for discussion and critical reading of the manuscript.
1. Dawood FS, Jain S, Finelli L, Shaw MW, Lindstrom S, Garten RJ, et al
. Emergence of a novel swine-origin influenza A (H1N1) virus in humans. N Engl J Med 2009; 360:2605–2615.
2. Garten RJ, Davis CT, Russell CA, Shu B, Lindstrom S, Balish A, et al
. Antigenic and genetic characteristics of swine-origin 2009 A(H1N1) influenza viruses circulating in humans. Science 2009; 325:197–201.
3. Cohen J, Enserink M. Swine flu. After delays, WHO agrees: the 2009 pandemic has begun. Science 2009; 324:1496–1497.
4. Hancock K, Veguilla V, Lu X, Zhong W, Butler EN, Sun H, et al
. Cross-reactive antibody responses to the 2009 pandemic H1N1 influenza virus. N Engl J Med 2009; 361:1945–1952.
5. Greenberg ME, Lai MH, Hartel GF, Wichems CH, Gittleson C, Bennet J, et al
. Response to a monovalent 2009 influenza A (H1N1) vaccine. N Engl J Med 2009; 361:2405–2413.
6. Clark TW, Pareek M, Hoschler K, Dillon H, Nicholson KG, Groth N, Stephenson I. Trial of 2009 influenza A (H1N1) monovalent MF59-adjuvanted vaccine. N Engl J Med 2009; 361:2424–2435.
7. 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.
8. Vajo Z, Tamas F, Sinka L, Jankovics I. Safety and immunogenicity of a 2009 pandemic influenza A H1N1 vaccine when administered alone or simultaneously with the seasonal influenza vaccine for the 2009-10 influenza season: a multicentre, randomised controlled trial. Lancet 2010; 375:49–55.
9. Plennevaux E, Sheldon E, Blatter M, Reeves-Hoche MK, Denis M. Immune response after a single vaccination against 2009 influenza A H1N1 in USA: a preliminary report of two randomised controlled phase 2 trials. Lancet 2010; 375:41–48.
10. Liang XF, Wang HQ, Wang JZ, Fang HH, Wu J, Zhu FC, et al
. Safety and immunogenicity of 2009 pandemic influenza A H1N1 vaccines in China: a multicentre, double-blind, randomised, placebo-controlled trial. Lancet 2010; 375:56–66.
11. Bickel M, Wieters I, Khaykin P, Nisius G, Haberl A, Stephan C, et al
. Low rate of seroconversion after vaccination with a split virion, adjuvanted pandemic H1N1 influenza vaccine in HIV-1-infected patients. AIDS 2010; 24:F31–F35.
12. Kroon FP, van Dissel JT, de Jong JC, Zwinderman K, van Furth R. Antibody response after influenza vaccination in HIV-infected individuals: a consecutive 3-year study. Vaccine 2000; 18:3040–3049.
13. Malaspina A, Moir S, Orsega SM, Vasquez J, Miller NJ, Donoghue ET, et al
. Compromised B cell responses to influenza vaccination in HIV-infected individuals. J Infect Dis 2005; 191:1442–1450.
14. 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.
15. 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 Vaccine Immunol 2008; 15:253–259.
16. Klein MB, Lu Y, DelBalso L, Cote S, Boivin G. Influenza virus infection is a primary cause of febrile respiratory illness in HIV-infected adults, despite vaccination. Clin Infect Dis 2007; 45:234–240.
18. Products. ECfPM. Note for guidance on harmonization of requirements for influenza vaccines (CPM/BWP/214/96)
. London: European Agency for Evaluation of Medical Products; 1997.
19. Moir S, Fauci AS. B cells in HIV infection and disease. Nat Rev Immunol 2009; 9:235–245.
20. Khurana S, Chearwae W, Castellino F, Manischewitz J, King LR, Honorkiewicz A, et al
. Vaccines with MF59 adjuvant expand the antibody repertoire to target protective sites of pandemic avian H5N1 influenza virus. Sci Transl Med 2010; 2:15ra15.
21. Fuller JD, Craven DE, Steger KA, Cox N, Heeren TC, Chernoff D. Influenza vaccination of human immunodeficiency virus (HIV)-infected adults: impact on plasma levels of HIV type 1 RNA and determinants of antibody response. Clin Infect Dis 1999; 28:541–547.
22. Morris L, Binley JM, Clas BA, Bonhoeffer S, Astill TP, Kost R, et al
. HIV-1 antigen-specific and -nonspecific B cell responses are sensitive to combination antiretroviral therapy. J Exp Med 1998; 188:233–245.
23. Beyer WE, Palache AM, Luchters G, Nauta J, Osterhaus AD. Seroprotection rate, mean fold increase, seroconversion rate: which parameter adequately expresses seroresponse to influenza vaccination? Virus Res 2004; 103:125–132.
24. Sasaki S, He XS, Holmes TH, Dekker CL, Kemble GW, Arvin AM, Greenberg HB. Influence of prior influenza vaccination on antibody and B-cell responses. PLoS One 2008; 3:e2975.
25. Fiore AE, Bridges CB, Cox NJ. Seasonal influenza vaccines. Curr Top Microbiol Immunol 2009; 333:43–82.
Keywords:© 2011 Lippincott Williams & Wilkins, Inc.
HIV-positive and HIV-negative individuals; MF-59 adjuvant; pandemic influenza vaccine