JAIDS Journal of Acquired Immune Deficiency Syndromes:
Anti-Influenza Serum and Mucosal Antibody Responses After Administration of Live Attenuated or Inactivated Influenza Vaccines to HIV-Infected Children
Weinberg, Adriana MD*; Song, Lin-Ye PhD†; Walker, Robert MD‡; Allende, Maria MD‡; Fenton, Terence EdD†; Patterson-Bartlett, Julie BS*; Nachman, Sharon MD§; Kemble, George PhD‡; Yi, Ting-Ting PhD‡; Defechereux, Patricia PhD‖; Wara, Diane MD‖; Read, Jennifer S MD, MS, MPH, DTM&H¶; Levin, Myron MD* ; for the IMPAACT P1057 Team
From the *Department of Pediatrics, University of Colorado Denver, Aurora, CO; †Harvard School of Public Health, Center for Biostatistics in AIDS Research, Boston, MA; ‡MedImmune, Inc, Gaithersburg, MD; §Department of Pediatric Infectious Diseases, Stony Brook, New York State University, Stony Brook, NY; ‖Department of Pediatrics, University of California, San Francisco, CA; and ¶Eunice Kennedy Shriver National Institute of Child Health and Human Development, Bethesda, MD.
Received for publication December 15, 2009; accepted March 10, 2010.
Supported by grant number 5 MO1-RR00069, General Clinical Research Centers Program, National Center for Research Resources, NIH; N01-HD-3-3162 and by grant number U01AI068632 and 1 U01 AI068616 from the National Institute of Allergy and Infectious Diseases; and N01-HD-3-3345 and contract N01-HD-33162 (AW) from the Eunice Kennedy Shriver National Institute of Child Health and Human Development; and by MedImmune LLC.
The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of Allergy and Infectious Diseases; the Eunice Kennedy Shriver National Institute of Child Health and Human Development; or the National Institutes of Health.
IMPAACT/PACTG P1057 Sites and Contributors are listed in Appendix I.
Correspondence to: Adriana Weinberg, MD, Professor of Pediatrics, Medicine and Pathology Director, Clinical Virology Laboratory University of Colorado Denver, Mail Stop 8604, 12700 East 19th Avenue, Room 11126, Aurora, CO 80045 (e-mail: firstname.lastname@example.org).
Background: Live-attenuated influenza vaccine (LAIV) prevents more cases of influenza in immune-competent children than the trivalent inactivated vaccine (TIV). We compared the antibody responses to LAIV or TIV in HIV-infected children.
Methods: Blood and saliva obtained at enrollment, 4 and 24 weeks postimmunization from 243 HIV-infected children randomly assigned to TIV or LAIV were analyzed.
Results: Both vaccines increased the anti-influenza neutralizing antibodies at 4 and 24 weeks postimmunization. At 4 weeks postimmunization, TIV recipients had 2-fold to 3-fold higher neutralizing antibody titers than LAIV recipients, but the proportions of subjects with protective titers (≥1:40) were similar between treatment groups (96%-100% for influenza A and 81%-88% for influenza B). Both vaccines increased salivary homotypic IgG antibodies, but not IgA antibodies. Both vaccines also increased serum heterosubtypic antibodies. Among HIV-specific characteristics, the baseline viral load correlated best with the antibody responses to either vaccine. We used LAIV-virus shedding as a surrogate of influenza infection. Influenza-specific humoral and mucosal antibody levels were significantly higher in nonshedders than in shedders.
Conclusions: LAIV and TIV generated homotypic and heterosubtypic humoral and mucosal antibody responses in HIV-infected children. High titers of humoral or mucosal antibodies correlated with protection against viral shedding.
Yearly immunization of HIV-infected individuals with the inactivated trivalent influenza vaccine (TIV) is recommended1 and is efficacious in these patients.2,3 Because antibody responses to TIV are not always adequate in this population,4-6 additional immunization of household contacts is strongly encouraged to further protect HIV-infected patients against influenza. A live attenuated influenza vaccine (LAIV) is licensed in the United States for immunization of healthy individuals. This vaccine is more effective than TIV in healthy children7-10; confers protection against infection with mismatched strains of influenza;11,12 and may provide a longer duration of protection than TIV.13,14 Furthermore, >50% of children <5 years of age immunized for the first time with LAIV achieve protective hemagglutination inhibition (HAI) titers ≥1:40 after the first dose of vaccine.15
We demonstrated that LAIV was safe and immunogenic in previously immunized HIV-infected children.16 Shedding of the vaccine strain influenza, which occurred in 23% of HIV-infected children, was limited to the first week after vaccination in accordance with the pattern observed in children without HIV infection.17 Antibody responses, measured by HAI, tended to be lower in LAIV compared with TIV recipients, which also was observed in uninfected children. However, protection conferred against influenza infection by LAIV, which is known not to correlate with HAI antibody titers, is presumed to depend on other immune mechanisms.18-24 An immune correlate of protection conferred by LAIV is highly desirable and is being actively pursued.
The goal of this study was to further characterize antibody responses to LAIV and TIV of HIV-infected children, including neutralizing antibodies, mucosal antibodies measured in saliva, and heterosubtypic HAI antibodies, which have not previously been described in this population. We also investigated for the first time the correlation of serum neutralizing antibodies and of mucosal Immunogloblin (Ig)G and IgA antibodies with shedding of LAIV viruses.
Study Population and Design
HIV-infected children and adolescents 5-18 years of age were randomly assigned in late September to late November of 2004 to receive LAIV (Arm A) or TIV (Arm B). Inclusion criteria were stable combination antiretroviral therapy for ≥16 weeks before immunization; plasma viral load <60,000 copies per milliliter; CD4 ≥15% within 60 days of enrollment; and immunization with TIV at least once in the 2 years preceding the study. Potential subjects were excluded if they received immunomodulatory therapy within 60 days before enrollment, inactivated or live vaccines within 14 and 30 days, respectively, of enrollment, and if they had any of the contraindications listed in the package insert of either vaccine. In each study arm the vaccinees were stratified by the following nadir CD4% [lowest CD4% before study entry and/or initiation of highly active antiretroviral therapy (HAART)] criteria: group 1 <15%; group 2 ≥ 15%, and <25%; and 3 ≥ 25%.
On study day 0, arm A (LAIV) received the frozen formulation of Influenza Virus Vaccine Live, “intranasal,” (FluMist; MedImmune, Gaithersburg, MD) 0.5 mL (0.25 mL per nostril); arm B (TIV) received Influenza Viral Vaccine, “intramuscular,” (Fluzone; Aventis Pasteur, Inc, Bridgewater, NJ) 0.5 mL in the deltoid muscle. The strains comprising the vaccines were those recommended by the US Public Health Service for the 2004/2005 season: A/New Caledonia/20/99 (H1N1); A/Wyoming/3/2003 (H3N2) [an A/Fujian/411/2002-like virus]; and B/Jilin/20/2003 (LAIV) or B/Jiangsu/10/2003 (TIV); both are B/Shanghai/361/2002-like viruses.
Subjects had blood and saliva collected for antibody measurements at study day 0, and weeks 4 and 24. Saliva was collected using OraSure kits as per manufacturer's instructions. LAIV recipients also had viral shedding from the nares monitored on days 3, 14, and 28 using viral culture and polymerase chain reaction, as previously described.16
Influenza-Specific HAI Assay (University of Colorado)
Serum samples were treated with receptor-destroying enzyme from Vibrio cholerae (Denka-Seiken, Campbell, CA). These were diluted 1:10 in saline, and subsequent serial 2-fold dilutions of the sera were used in a standard HAI assay using 4 hemagglutinating units of the viruses or antigen and 0.75% guinea pig red blood cells. Serum samples with titers ≥10 and ≥40 were considered indicative of immune responses and protection, respectively. The antigens used in the assays were: A/New Caledonia/20/99 (H1N1), A/Wyoming/03/2003 (H3N2), A/Sydney/05/97 (H3N2) and B/Yamanashi/166/98 cold-adapted viruses, and B/Yamanashi/166/98 (Shanghai-like) antigen generously provided by Dr. Alexander Klimov at the Centers for Disease Control and Prevention.
Influenza-Specific Neutralizing Antibody Assay (MedImmune)
An influenza microneutralization (MN) assay was used to determine specific influenza-neutralizing antibody titers in serum. Serial dilutions of receptor-destroying enzyme-treated serum were made in duplicate in 96-well plates, and influenza virus at pre-established concentration was added to the serum-containing wells. After incubation for 1 hour at 33°C, the serum-virus mixtures were inoculated onto washed confluent Mardin Darby canine kidney (MDCK) monolayers containing 2 × 105 cells per well in 96-well plates and incubated for 6 days at 33°C. After incubation, culture medium was removed, replaced with Alamar Blue (10% v/v in phosphate buffered saline), and the plates incubated at 33°C for a further 5 hours, after which the absorbance in individual wells was read (531 nm excitatory wavelength and 590 nm emission wavelength) on a Perkin-Elmer Multi Label Counter. Absence of infection and associated cytopathic effect was indicated by the ability of intact cells to take up and metabolize Alamar Blue with positive absorbance in the corresponding wells. Conversely, infection of cells and consequent cytopathic effect resulted in an inability to metabolize Alamar Blue and absence of absorbance. The neutralizing titer was defined as the reciprocal of the serum dilution in the last well showing evidence of Alamar Blue metabolism. A 4-fold or greater difference in titer between 2 sera was considered significant.
Influenza-Specific Salivary Antibodies
Influenza strain-specific IgG and IgA were assessed by kinetic enzyme-linked immunosorbent assay (ELISA). Microtiter plates (Costar, Lowell, MA) were coated with Fluzone at 1.56 ng/mL in carbonate buffer overnight at 4°C. After washing and blocking, serial dilutions of saliva in boric acid solution were added to triplicate wells. Plates were washed, incubated with biotinylated goat anti-human IgG or IgA (Biosource International, Carlsbad, CA), followed by streptavidin-peroxidase (Sigma, St. Louis, MO) and TMB substrate. The absorbance of the test wells, measured with a Microplate Reader (Molecular Devices, Sunnyvale, CA), was interpolated onto a standard curve using a pretitered control saliva sample. Results were divided by the total amount of IgG or secretory IgA in the saliva sample measured by ELISA (ALPCO Diagnostics, Salem, NH). Results are expressed as influenza-specific ELISA units/IgG or IgA concentration in saliva.
Paired t tests or Wilcoxon matched pairs signed ranks tests were used to compare baseline with postimmunization responses. Comparisons across groups that differed by treatment, HIV-specific characteristics, or viral shedding used F tests, Wilcoxon rank sum tests, or Fisher exact tests. For some of the analyses, the data were log10 transformed to normalize their distributions. For data, which included values of 0, a very small value (0.001) was added to enable log transformation. Correlation analyses consisted of Spearman or Pearson correlations and linear or logistic regressions with quantitative data for all predictors, except for plasma viral load, which was dichotomized as <400 or ≥400 copies per milliliter. Statistical significance was defined as an alpha level of P ≤ 0.05.
Demographic Characteristics of the Study Population
The study randomly assigned 243 HIV-infected children to LAIV or TIV treatment arms. The demographic characteristics were similar in the 2 arms (Table 1). The mean age at enrollment was 12 years. More than 70% of the participants had plasma HIV RNA concentrations (viral load) <400 copies per milliliter and CD4% >25 at baseline. However, nadir CD4%, before study entry, varied between 0.4% and 41% with a median of 17%. All children were on treatment containing ≥1 nucleoside reverse transcriptase inhibitors. In addition, 22% of the antiretroviral regimens included both nonnucleotide reverse transcriptase inhibitors and protease inhibitors, 60% only protease inhibitors and 17% only nonnucleotide reverse transcriptase inhibitors.
MN Responses to LAIV and TIV in HIV-Infected Children on HAART
The baseline MN titers for A H1N1 New Caledonia, A H3N2 Wyoming and B Jilin were similar in the 2 treatment arms (Fig. 1). At 4 and 24 weeks after immunization, both TIV and LAIV recipients showed significant increases in MN titers against all strains in the vaccines (P ≤ 0.02), but the week 4 MN titers were higher among TIV compared with LAIV recipients (P ≤ 0.002).
An MN titer ≥1:40 has been associated with ≥50% reduction in influenza infection and morbidity in children.25 Table 2 shows that both treatment groups had similar proportions of subjects with MN titers ≥1:40 at baseline, week 4 and week 24 for each of the 3 strains in the vaccines.
Analyses to identify correlates of the magnitude of the MN response to TIV and LAIV in HIV-infected children on HAART (Table 3) showed the strongest correlation with baseline MN titers (P < 0.0001). The multivariate analysis including baseline MN titers, plasma viral load, CD4%, CD8%, and CD19% showed that among HIV-specific immunologic and virologic parameters, only the plasma viral load at baseline was independently associated with responses to all 3 viruses in TIV recipients and to A H1N1 New Caledonia in LAIV recipients.
Mucosal Antibody Responses to LAIV and TIV in HIV-Infected Children on HAART
Figure 2 shows the changes in influenza-specific salivary IgG after vaccination. The salivary responses to the vaccines followed the same pattern as the MN responses. In addition, salivary influenza-IgG concentrations were significantly associated with serum antibody titers against influenza A H3N2 Wyoming at all time points (P < 0.0001). The multivariate analysis showed that week 4 salivary anti-influenza-IgG responses to either vaccine were independently associated with baseline concentrations and with baseline plasma HIV viral load (Table 4).
Influenza-specific salivary IgA were detected only in a minority of study participants. There were no significant increases in salivary influenza-IgA at 4 weeks after LAIV or TIV administration. At 24 weeks after vaccination, salivary influenza-IgA concentrations were significantly higher compared with baseline in TIV recipients (P < 0.01) but not in LAIV recipients (P = 0.17). Salivary influenza-IgA concentrations were weakly correlated with influenza A H3N2 serum antibody titers and with salivary influenza-IgG concentrations at baseline (rho of 0.184 and 0.146, respectively; P ≤ 0.03 for both). Week 4 salivary influenza-IgA responses to either vaccine were significantly associated with baseline salivary influenza-IgA concentrations. In TIV recipients, week 4 salivary influenza-IgA concentrations also were associated with week 4 serum antibody titers (rho = 0.242, P = 0.01) and weakly associated with salivary influenza-IgG concentrations (rho = 0.166, P = 0.09). In either treatment arm, there were no significant associations of salivary influenza-IgA concentrations with baseline plasma viral load, CD4%, CD8%, or CD19%.
Immunecorrelates of Protection Against Shedding of LAIV-Contained Viruses
To identify immune defenses associated with protection against influenza infection in HIV-infected children on HAART, we used shedding of the vaccine virus as a surrogate of infection (Fig. 3). There were 113 children with baseline antibody and vaccine virus excretion data. Of these, 22 children shed A H1N1 New Caledonia, 9 B Jilin, and 3 A H3N2 Wyoming vaccine viruses during the first week after immunization. Children who shed A H1N1 New Caledonia had significantly lower HAI and MN titers (P < 0.001 for both) than children without shedding. The comparison of subjects who shed B Jilin showed similar trends, but due to the smaller number of subjects who shed B Jilin, the MN or HAI titer differences between shedders and nonshedders did not reach statistical significance. An analysis of the factors associated with protection against shedding of A H3N2 Wyoming vaccine virus was not performed due to the small number of shedders. The comparison of influenza-IgG and IgA between shedders of any influenza vaccine strain and nonshedders showed increased concentrations of both types of antibodies in nonshedders (P = 0.05 and 0.02, respectively).
HAI and MN titers ≥1:40 are considered predictors of protection against influenza infection and morbidity in children. To test the ability of these thresholds to predict protection against viruses contained in LAIV, we compared the proportions of shedders and nonshedders with antibody titers higher than the above listed thresholds (Table 5). Neither HAI nor MN titers ≥1:40 were completely protective against vaccine virus shedding. Influenza A H1N1 shedders had significantly lower proportions of subjects with protective HAI or MN titers compared with nonshedders (P ≤ 0.01). There was a trend toward lower prevalence of MN titers ≥1:40 among B Jilin shedders compared with nonshedders (P = 0.06) but no difference in the proportions of subjects with HAI titers ≥1:40. There were no significant differences in MN or HAI titers between shedders and nonshedders of A H3N2 Wyoming.
Heterosubtypic HAI Responses to LAIV and TIV in HIV-Infected Children
At 4 weeks after vaccination, HAI antibody titers were measured against A H3N2 Sydney and B Yamanashi, which were not included in the 2004/2005 influenza vaccines. Both LAIV and TIV recipients showed increases in antibody titers to these mismatched influenza strains (Table 5). Although baseline HAI titers against the mismatched strains were similar in the 2 treatment groups, TIV recipients had significantly higher HAI titers compared with LAIV recipients against either mismatched influenza virus at 4 and 24 weeks after vaccination.
Both LAIV and TIV administration increased influenza-specific MN titers in HIV-infected children on HAART. The elevated MN titers persisted for at least 6 months, suggesting that the influenza-specific seroprotection conferred by each vaccine to HIV-infected children on HAART lasts through an entire influenza season. Although compared with LAIV, TIV administration resulted in significantly higher MN titers against 2 of the 3 influenza serotypes in the vaccine, the proportions of subjects with postimmunization influenza MN titers ≥1:40, the threshold associated with protection against infection and morbidity in HIV-uninfected children, was similar among LAIV and TIV recipients for all the viruses in the vaccines. In addition, we used shedding of the LAIV strains to validate the protective value of MN titers >1:40 in HIV-infected children.
The MN responses to the influenza vaccines of HIV-infected children on HAART were strongly associated with baseline MN titers, which negatively correlated with baseline plasma viral load and CD8% and positively correlated with baseline CD4%. Furthermore, a multivariate analysis showed that the week 4 response to all 3 viruses in TIV and to 1 of the viruses in LAIV was also independently associated with baseline plasma viral load. Taken together, these findings underscore the importance of controlling viral replication and preserving CD4 cells for the development of protective immune responses to influenza vaccines and wild-type infection.
Because influenza infection is acquired through colonization of the respiratory tract, mucosal immunity is deemed to play an important role in protection against influenza infection. Using saliva as a surrogate sample for mucosal immunity, we showed that both TIV and LAIV increased the salivary influenza-specific IgG titers. Influenza-specific IgA antibody was present only in a minority of vaccinees at baseline and did not increase significantly at 4 weeks after immunization. At 24 weeks after immunization, the influenza-specific IgA levels in saliva were significantly higher compared with the baseline levels in LAIV recipients but not in TIV recipients. The limited advantage of LAIV over TIV with respect to mucosal IgA antibody responses was surprising because LAIV is administrated intranasally, and we expected it to stimulate higher local antibody production compared with TIV. The extent to which the antibody titers in the saliva reflect the antibody production in the respiratory tract has been debated. In this study, we showed a significant association between the influenza-specific IgA and IgG concentrations in saliva and protection against shedding of LAIV viruses. Because LAIV is administered intranasally, we interpreted these results as an indication that salivary influenzas-specific antibody correlates with functional nasal influenza-specific antibody.
Influenza-specific salivary IgG antibodies were highly correlated with MN titers both before and after immunization with either vaccine. Furthermore, the same immunologic and virologic parameters that affected the MN titers also determined the influenza-specific salivary concentrations. Taken together, these findings suggested that the salivary influenza-specific IgG may represent a transudate, although local influenza-specific IgG production could not be ruled out. In contrast to IgG, neither baseline nor week 4 influenza-specific salivary IgA correlated with serum HAI or MN (not shown) titers, suggesting that the IgA was the result of local production. Influenza-specific salivary IgA responses to vaccination correlated with baseline influenza-specific IgA concentrations but did not correlate with any of the HIV-specific immunologic or virologic parameters. The latter findings have to be interpreted with caution because most subjects did not have detectable levels of influenza-specific IgA in the saliva, which may have weakened the ability to detect any associations.
The influenza serotypes in the seasonal vaccines are not always well matched to the serotypes that circulate during the influenza season. Compared with TIV, LAIV administration confers increased protection against mismatched influenza serotypes in children,8,11 but the opposite phenomenon was suggested in adults.26,27 We measured the heterosubtypic HAI antibody responses to LAIV and TIV in HIV-infected children against influenza viruses that circulated several years before this study was performed. Administration of either vaccine was followed by an increase of the HAI antibody titers to heterosubtypic viruses, which were in fact highly correlated with the HAI titers to the viruses in the vaccines, suggesting cross reactivity at the level of antibodies or memory B cells.
This study showed that HIV-infected children on HAART respond to both LAIV and TIV with MN, mucosal, and heterosubtypic antibody production. As previously demonstrated in healthy children19,28,29, antibody titers tended to be higher after TIV compared with LAIV administration. However, the proportion of HIV-infected children that achieved MN antibody titers considered protective against influenza were similar among LAIV and TIV recipients. Both humoral and mucosal antibody titers were associated with protection against shedding of LAIV viruses, suggesting that antibodies in both compartments may contribute to the protection against infections caused by influenza viruses in general.
We thank Nancy Tustin, Cristina Gonzaga, and Howard Gutzman for technical and data management support.
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APPENDIX I: IMPAACT/PACTG P1057 Sites and Contributors
Site 3701 Johns Hopkins University Hospital (Beth Griffin, RN; Nancy Hutton, MD; Mary Joyner, NP; Andrea Ruff, MD); 4701 Duke University-Pediatric (Joan Wilson, RN; Mary Jo Hassett, RN; Carole Mathison; John Swetnam); 5006 Harlem Hospital (Elaine Abrams, MD; Maxine Frere, RN; LisaGaye Robinson, MD); 5012 NYU Medical Center/Bellevue (William Borkowsky, MD; Sandra Deygoo, BS; Siham Akleh, RN; Aditya Kaul, MD); 5018 University of South Florida Physicians Group (Jorge Lujan-Zilberman, MD; Patricia Emmanuel, MD; Carolyn Graisbery, RN; Carina Rodriguez, MD); 5024 Children's Hospital Kings and Daughters (Randall G. Fisher, MD; Kenji M. Cunnion, MD, MPH; Laura Sass, MD; Donna Sandifer, RN); 5026 Mount Sinai (Mary S. Dolan, RN; Roberto Posada, MD); 5038 Yale University School of Medicine (Warren A. Andiman, MD; Leslie Hurst, BS; Sostena Romano, APRN, MBA); 5040 SUNY Health Science Center (Denise Ferraro, RN; Michele Kelly, PNP; Margaret Oliver, LPN); 5051 University of Florida Health Science Center (Mobeen Rathore, MD; Nizar Maraqa, MD; Kathy Thomas, MA; Angela Lala, LPN); 5052 The Children's Hospital- U. of Colorado; (Mark Abzug MD; Emily Barr; Megan Canon; Josephine Greenquist); 5057 University of Rochester-Pediatric Component (Geoffrey A. Weinberg, MD; Francis Gigliotti, MD; Barbra Murante, RNC, PNP; Susan Laverty, RN); 5095 Tulane University (Margarita Silio, MD; Thomas Alchediak, MD; Cheryl Borne, RN; Sheila Bradford, RN); 6701 The Children's Hospital of Philadelphia IMPAACT CTU (Steven D. Douglas, MD; Richard M. Rutstein, MD; Carol A. Vincent, CRNP, MSN; Patricia C. Coburn, RN, BSN); 7301 University of Massachusetts Medical School (Katherine Luzuriaga, MD; Richard Moriarty, MD; William (Jerry) Durbin, MD; Donna Picard, RN); 60336 Baylor College of Medicine (Chivon D. McMullen-Jackson, RN, ADN; Theresa Aldape, LMSW; Mary E. Paul, MD; Heidi L. Schwarzwald, MD, MPH); 60422 St. Jude/Memphis (Gregory Storch, MD; Laura Pickering, RN; Katherine Knapp, MD; Jill Utech, RN); 60444 Family Clinical Trials Center (Mavis Dummitt, RN; Caroline Nubel; Stefan Hagmann, MD; Murli Purswani, MD); 2901 Boston Children's Hospital; 3601 UCLA Medical Center; 4001 Children's Hospital of Chicago; 4501 UCSF Medical Center; 4601 UCSD Medical Center; 5008 Children's Hospital at SUNY Downstate; 5013 Jacobi Medical Center; 5031 City Hospital at San Juan; 5041 Children's Hospital of Michigan; 5055 Children's Diagnostic & Treatment Center of South Florida; 5056 University of Florida at Gainesville; 6501 St. Jude Children's Research Hospital; 7701 University of Alabama, Birmingham; 60341 Columbia Collaborative-HIV/AIDS; 60349 University of Miami Ped. Perinatal HIV/AIDS; 60358 NJ Medical School. Cited Here...
antibodies; HIV-infected children; influenza vaccines; viral shedding
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