Standard Trivalent Influenza Virus Protein Vaccination Does Not Prime Antibody-Dependent Cellular Cytotoxicity in Macaques

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   Fig 1

   Timeline of TIV vaccination and H1N1/H3N2 challenge in pigtail macaques.
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   Fig 2

   Influenza virus-specific binding antibodies following vaccination and infection of macaques. (A to D) We used either purified H1N1 (A/California/07/2009) (A and B) or H3N2 (A/Perth/16/2009) (C and D) virus to measure binding antibodies in plasma samples taken from 6 macaques prior to immunization (Pre-TIV), 4 weeks postimmunization with two doses of TIV (Post-TIV), 4 weeks post-H1N1 challenge (Post-H1N1), and 4 weeks post-H3N2 challenge (Post-H3N2) by ELISA. (E and F) We used purified H1N1 (A/California/07/2009) to measure binding antibodies in plasma samples taken from 6 macaques prior to H1N1 (Pre-H1N1) challenge, post-H1N1 challenge (Post-H1N1), and post-H3N2 challenge (Post-H3N2) by ELISA. Plasma samples were all titrated via half-log dilutions, with a starting dilution of 1:100. The antibody titers are given as the reciprocal of the dilution giving an OD reading four times background. Antibody titers were compared for animals between time points, and statistical analysis was performed by Friedman test, followed by a separate Wilcoxon test (B, D, and F). The horizontal lines represent the median values of groups.
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   Fig 3

   H1-specific ADCC after vaccination and H1N1 challenge. (A) Flow cytometry plots of NK cell activation ADCC assays. Gating strategy is shown in the upper plots. The lower plots show representative examples of antibody-mediated activation of CD3⁻ NKG2A⁺ NK cells expressing IFN-γ and CD107a following exposure of healthy donor macaque PBMCs to either (i) no H1 protein (unstimulated) with post-H1N1 infection plasma, (ii) H1 protein and plasma postvaccination/preinfection, or (iii) H1 protein and post-H1N1 infection plasma. (B to G) Frequencies of NK cells expressing either IFN-γ or CD107a in the presence of plasma from 6 macaques prevaccination (B and C), 4 weeks after 2 TIV vaccinations (D and E), and 4 weeks after H1N1 infection (F and G). ADCC responses to the recombinant HA proteins from H1N1 influenza viruses A/California/7/2009 (CA/09), A/Solomon Islands/03/2006 (SI/06), A/New Caledonia/20/1999 (NC/99), and A/Brisbane/59/2007 (BR/07) were measured. The horizontal lines represent the median values of groups. Statistical analysis was performed by Friedman test, followed by a separate Wilcoxon matched-pairs signed-rank test. All samples were corrected for background based on their responses to wells containing plasma but with no plate-bound antigen.
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   Fig 4

   ADCC to H1 protein in unvaccinated macaques versus vaccinated macaques after H1N1 challenge. (A and B) Frequencies of NK cells expressing IFN-γ and CD107a in response to plasma from 6 control unvaccinated macaques prior to H1N1 infection. (C and D) Comparison of the frequencies of NK cells expressing IFN-γ and CD107a in response to plasma from either 6 control unvaccinated macaques (squares) or 6 vaccinated macaques (circles) 4 weeks after H1N1 infection. (E and F). Comparison of the frequencies of NK cells expressing IFN-γ and CD107a toward recombinant HA protein from A/Solomon Islands/3/2006 in the presence of plasma from either 6 control unvaccinated macaques (open symbols) or 6 vaccinated macaques (solid symbols) at days 0, 2, 4, 8, 15, and 29 post-H1N1 infection. ADCC responses to the recombinant HA proteins from H1N1 influenza viruses A/California/7/2009 (CA/09), A/Solomon Islands/3/2006 (SI/06), A/New Caledonia/20/1999 (NC/99), and A/Brisbane/59/2007 (BR/07) were measured. The horizontal lines represent the median values of groups. Statistical analysis was performed by separate Mann-Whitney U tests. All samples were corrected for background based on their responses to wells containing plasma but with no plate-bound antigen.
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   Fig 5

   Lack of ADCC cross-recognition of other HA subtypes after H1N1 infection of macaques. Comparison of the frequencies of NK cells expressing IFN-γ (A) and CD107a (B) in response to plasma from either 6 unvaccinated macaques (squares) or 6 vaccinated macaques (circles) 4 weeks after H1N1 infection. ADCC responses to the recombinant HA proteins from H1N1 (A/California/7/2009), H2N2 (A/Japan/305/1957), H3N2 (A/Brisbane/10/2007), H4N6 (A/Swine/Ontario/01911-1/1999), H5N1 (A/Vietnam/1203/2004), and recombinant NP (A/Puerto Rico/8/1934) were measured. The horizontal lines represent the median values of groups. Statistical analysis was performed by separate Mann-Whitney U tests. All samples were corrected for background based on their responses to wells containing plasma but with no plate-bound antigen.
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   Fig 6

   ADCC responses to H3N2 HA proteins in unvaccinated macaques compared to vaccinated H1N1-infected macaques and H1N1-infected macaques after H3N2 challenge. (A and B) Frequencies of NK cells expressing IFN-γ and CD107a in response to plasma from 6 unvaccinated/H1N1 challenge macaques (circles), 6 H1N1-infected macaques (squares), and 6 naive control macaques (triangles) prior to H3N2 challenge. (C and D) Frequencies of NK cells expressing IFN-γ and CD107a in response to plasma from 6 unvaccinated/H1N1 challenge macaques (circles), 6 H1N1-infected macaques (squares), and 6 naive control macaques (triangles) 4 weeks after H3N2 infection. (E and F) Frequencies of NK cells expressing IFN-γ and CD107a in the presence of plasma from 6 unvaccinated/H1N1 challenge macaques (circles), 6 H1N1-infected macaques (squares), and 6 naive control macaques (triangles) to recombinant HA proteins from seasonal H3N2 strains A/Wyoming/03/2003 (WY/06), A/Aichi/2/1968 (AI/68), and A/Brisbane/10/2007 (BR/07) 4 weeks after H3N2 infection. ADCC responses to the recombinant HA proteins from seasonal H3N2 and H1N1 influenza viruses A/Solomon Islands/03/2006 (SI/06) and A/Wyoming/03/2003 were also measured. The horizontal lines represent the median values of groups. Statistical analysis was performed by 2 separate Kruskal-Wallis tests, followed by separate Mann-Whitney U tests. All samples were corrected for background based on their responses to wells containing plasma but with no plate-bound antigen.
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   Fig 7

   Influenza virus-specific CD8 T cell responses over time following TIV vaccination and influenza virus infection of macaques. (A) Representative plots of Mane-A1*08401–RA9 tetramer-positive CD3⁻ CD8⁺ T cells from a vaccinated H1N1- and H3N2-infected macaque (animal C3752) at week 0 prior to TIV vaccination, week 2 post-second TIV vaccination, week 1 post-H1N1 infection, and week 1 post-H3N2 infection. (B) Frequencies of RA9-specific T cells in 6 vaccinated H1N1-H3N2 macaques (TIV), 6 unvaccinated H1N1-H3N2 macaques (H1N1), and 6 H3N2 macaques (H3N2) at time points prior to TIV vaccination (Fig. 1 shows the groups), 4 weeks after the final TIV vaccination/pre-H1N1 infection, 1 week post-H1N1 infection (Post-H1N1), pre-H3N2 infection, and 2 weeks post-H3N2 infection (Post-H3N2). The horizontal lines represent the median values of groups. (C) Time course of median frequencies of RA9-specific CD8 T cells from 6 vaccinated H1N1-H3N2 macaques (TIV), 6 unvaccinated H1N1-H3N2 macaques (H1N1), and 6 H3N2 macaques (H3N2).

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