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%.
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.
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.