Browse Subject Areas

Click through the PLOS taxonomy to find articles in your field.

For more information about PLOS Subject Areas, click here.

  • Loading metrics

Determinants of Vaccine Immunogenicity in HIV-Infected Pregnant Women: Analysis of B and T Cell Responses to Pandemic H1N1 Monovalent Vaccine

  • Adriana Weinberg ,

    Affiliation University of Colorado Anschutz Medical Center, Aurora, Colorado, United States of America

  • Petronella Muresan,

    Affiliation Statistical and Data Analysis Center, Center for Biostatistics in AIDS Research, Harvard School of Public Health, Boston, Massachusetts, United States of America

  • Kelly M. Richardson,

    Affiliation University of Colorado Anschutz Medical Center, Aurora, Colorado, United States of America

  • Terence Fenton,

    Affiliation Statistical and Data Analysis Center, Center for Biostatistics in AIDS Research, Harvard School of Public Health, Boston, Massachusetts, United States of America

  • Teresa Dominguez,

    Affiliation University of Colorado Anschutz Medical Center, Aurora, Colorado, United States of America

  • Anthony Bloom,

    Affiliation Frontier Science and Technology Research Foundation, Buffalo, New York, United States of America

  • D. Heather Watts,

    Current address: Office of the U.S. Global AIDS Coordinator, U.S. Dept. of State, Washington, D.C., United States of America

    Affiliation Maternal and Pediatric Infectious Disease Branch, Eunice Kennedy Shriver National Institute of Child Health and Human Development, Bethesda, Maryland, United States of America

  • Mark J. Abzug ,

    Contributed equally to this work with: Mark J. Abzug, Sharon A. Nachman, Myron J. Levin

    Affiliation University of Colorado Anschutz Medical Center, Aurora, Colorado, United States of America

  • Sharon A. Nachman ,

    Contributed equally to this work with: Mark J. Abzug, Sharon A. Nachman, Myron J. Levin

    Affiliation State University of New York Health Science Center at Stony Brook, Stony Brook, New York, United States of America

  • Myron J. Levin ,

    Contributed equally to this work with: Mark J. Abzug, Sharon A. Nachman, Myron J. Levin

    Affiliation University of Colorado Anschutz Medical Center, Aurora, Colorado, United States of America

  • for the P1086 team

    Membership of the P1086 team is provided in the Acknowledgments.

Determinants of Vaccine Immunogenicity in HIV-Infected Pregnant Women: Analysis of B and T Cell Responses to Pandemic H1N1 Monovalent Vaccine

  • Adriana Weinberg, 
  • Petronella Muresan, 
  • Kelly M. Richardson, 
  • Terence Fenton, 
  • Teresa Dominguez, 
  • Anthony Bloom, 
  • D. Heather Watts, 
  • Mark J. Abzug, 
  • Sharon A. Nachman, 
  • Myron J. Levin


Influenza infections have high frequency and morbidity in HIV-infected pregnant women, underscoring the importance of vaccine-conferred protection. To identify the factors that determine vaccine immunogenicity in this group, we characterized the relationship of B- and T-cell responses to pandemic H1N1 (pH1N1) vaccine with HIV-associated immunologic and virologic characteristics.

pH1N1 and seasonal-H1N1 (sH1N1) antibodies were measured in 119 HIV-infected pregnant women after two double-strength pH1N1 vaccine doses. pH1N1-IgG and IgA B-cell FluoroSpot, pH1N1- and sH1N1-interferon γ (IFNγ) and granzyme B (GrB) T-cell FluoroSpot, and flow cytometric characterization of B- and T-cell subsets were performed in 57 subjects.

pH1N1-antibodies increased after vaccination, but less than previously described in healthy adults. pH1N1-IgG memory B cells (Bmem) increased, IFNγ-effector T-cells (Teff) decreased, and IgA Bmem and GrB Teff did not change. pH1N1-antibodies and Teff were significantly correlated with each other and with sH1N1-HAI and Teff, respectively, before and after vaccination. pH1N1-antibody responses to the vaccine significantly increased with high proportions of CD4+, low CD8+ and low CD8+HLADR+CD38+ activated (Tact) cells. pH1N1-IgG Bmem responses increased with high proportions of CD19+CD27+CD21- activated B cells (Bact), high CD8+CD39+ regulatory T cells (Treg), and low CD19+CD27-CD21- exhausted B cells (Bexhaust). IFNγ-Teff responses increased with low HIV plasma RNA, CD8+HLADR+CD38+ Tact, CD4+FoxP3+ Treg and CD19+IL10+ Breg.

In conclusion, pre-existing antibody and Teff responses to sH1N1 were associated with increased responses to pH1N1 vaccination in HIV-infected pregnant women suggesting an important role for heterosubtypic immunologic memory. High CD4+% T cells were associated with increased, whereas high HIV replication, Tact and Bexhaust were associated with decreased vaccine immunogenicity. High Treg increased antibody responses but decreased Teff responses to the vaccine. The proportions of immature and transitional B cells did not affect the responses to vaccine. Increased Bact were associated with high Bmem responses to the vaccine.


Pregnant women in their 2nd and 3rd trimesters and the first 2 weeks post-partum have a 3.3- to 5.5-fold greater risk of hospitalization for influenza-associated acute cardio-respiratory illness compared to non-pregnant women[16]. Additionally, influenza respiratory illness during pregnancy may increase the risk of premature delivery, fetal distress and emergency caesarean sections[7,8]. Heightened susceptibility to severe influenza illness during pregnancy is particularly evident during influenza pandemics as was observed during the pandemic caused by the pandemic influenza A H1N1 2009 (pH1N1)[13,5]. Vaccination is the most effective modality to combat the morbidity of influenza infections[9,10]. Administration of seasonal trivalent inactivated vaccines (IIV3) to pregnant women prevents severe infections in women and their infants up to 6 months of life and decreases premature deliveries[1016]. Although early studies showed that IIV3 had similar immunogenicity in pregnant women and non-pregnant adults[17], this concept was recently challenged[18,19].

HIV-infected adults do not seem to have greater influenza-associated morbidity than same-age uninfected controls except for those with CD4+ cells <200 cells/μL[2026]. This conclusion is uncertain with respect to HIV-infected pregnant women in whom the immunosuppressive effect of pregnancy may synergize with that of HIV infection. Furthermore, the immunogenicity of influenza vaccines is much lower in HIV-infected individuals compared with uninfected controls of the same age. We previously showed that HIV-infected pregnant women had lower hemagglutination inhibition (HAI) antibodies and cell-mediated immunity (CMI) in response to IIV3 compared with uninfected pregnant women[27]. Since low CD4+ cell numbers have been associated with poor responses to vaccines in HIV-infected individuals [2837], it is noteworthy that HIV-infected pregnant women experience a decrease of approximately 100 CD4+ cells/μL during pregnancy. This is particularly relevant, since the efficacy of IIV is predicated on its ability to generate HAI titers ≥1:40. This was based on the early observation that healthy young adults with HAI titers ≥1:40 had a 50% decrease in influenza disease[38]. Although this immune correlate with protection has recently been challenged[39], it continues to be used as a benchmark for evaluating the immunogenicity of influenza vaccines. Currently, the immune correlates of protection against influenza infection in HIV-infected individuals are not known and the mechanisms responsible for their poor antibody responses to IIV are also not well understood.

Antibody responses to influenza vaccines are T-cell dependent and, therefore, are affected by the functionality of T helper 1 (Th1) [40] and T follicular helper (Tfh) cells [41]. Both Th1 and Tfh functions are severely compromised in HIV-infected individuals and may contribute to the low immunogenicity of influenza vaccines [4244]. In addition, multiple B-cell abnormalities have been identified in HIV-infected individuals [45], which may also play a role in the poor antibody responses to vaccines. Although HIV does not replicate in B cells, it interferes with B-cell function through multiple interactions: gp120 with cellular DC-SIGN; CD40L incorporated into the virion membrane with cellular CD40; and complement fixing HIV antigen-antibody complexes with cellular CD21 [4652]. In addition, HIV Nef protein can be delivered to the B cells through immunologic synapses with CD4+ T cells and/or macrophages and impede the NFkB pathway, while also activating the suppressor of cytokine signaling (SOCS) pathway [49]. Additional indirect effects of HIV on B cells result from inflammation and lymphopenia. These ultimately translate into impaired immunoglobulin class switch recombination, loss of resting memory B cells (CD21+CD27+), abnormally high proportions of immature (CD10+) and activated (CD21-CD27+, CD95+ and/or CD38+) B cells, and increased B-cell turn-over and apoptosis [49,5355]. All these factors may contribute to the decreased antibody responses to vaccines [48,5659]. Furthermore, only some of the B-cell abnormalities are averted by lack of disease progression or reversed with HAART [60,61]. To effectively target efforts to improve vaccine responses in HIV-infected individuals, it is important to understand the relative contributions of each of these factors to decreased antibody responses to vaccines.

The goal of this study was to identify the factors associated with the immunogenicity of IIV in HIV-infected pregnant women. We previously reported that HIV-infected pregnant women enrolled in the P1086 study of the International Maternal Pediatric and Adolescent AIDS Clinical Trials group (IMPAACT) who received two 30μg-doses of pH1N1 monovalent vaccine (IIV1) had lower antibody responses than uninfected historical controls after a single 15μg-dose of the vaccine[62]. Here we evaluated the primary and anamnestic HAI, effector T-cell (Teff) and memory B-cell (Bmem) responses to pH1N1 IIV1 among P1086 participants and the relationships between these endpoints with B- and T-cell phenotypic characteristics. The relationship between immune responses to pH1N1 vaccine and pre-existing immunity to seasonal H1N1 (sH1N1) virus was also studied.

Materials and Methods

Study design

P1086 involved human subject research and was approved by the Colorado Multiple Institutional Review Board on 9/25/09; approval number 09–0803. Informed written consent was obtained from all participants. HIV-infected women 18 to 39 years of age, 14 to 34 weeks gestation, and on antiretroviral therapy, received two 30 μg doses of unadjuvanted, inactivated pH1N1 vaccine 21 to 28 days apart at 31 U.S. IMPAACT sites as previously described[62]. Serum was collected at entry, before administration of the 1st dose of vaccine; before administration of the 2nd dose (21 to 28 days post-dose 1); and 10 to 14 days post-dose 2. The 21 to 28 days post-dose 1 and 10 to 14 days post-dose 2 time points were selected to coincide with the anticipated peak primary and anamnestic antibody responses, respectively. Peripheral blood mononuclear cells (PBMCs) were isolated and cryopreserved for viability at the same time points.

Antibody responses measured by HAI

pH1N1 and sH1N1 HAI titers were measured as previously described [13] using A/California/7/2009 Pandemic X-179A and A/South Dakota/6/2007 H1N1 influenza viruses, respectively, obtained through a generous gift of Dr. A Klimov from the Centers of Disease Control. HAI titers were expressed as the reciprocal of the endpoint titer. Seroprotection was defined as a titer ≥ 40.

IFNγ and Granzyme B (GrB) CMI assays

PBMCs were cryopreserved according to a standardized protocol ( at the local laboratories, which were previously certified for PBMC cryopreservation; stored at ≤-150°C; and shipped in liquid nitrogen dry shippers to the testing lab at University of Colorado Anschutz Medical Campus. Cells were thawed slowly as previously described.[63] FluoroSpot assays were performed using commercial dual color IFNγ/Granzyme B FluoroSpot kits (MabTech) per manufacturer’s instructions with modifications. Thawed PBMCs were maintained overnight at 106 PBMC/mL in RPMI 1640 with glutamine (Gibco), 10% human AB serum (Gibco), 1% penicillin and streptomycin, and 1% Hepes buffer (Corning Cellgrow) at 37°C in a humidified 5% CO2 atmosphere. PBMCs with viability <70%, as measured with a Guava easyCyte 8HT instrument (Millipore), were excluded from the subsequent steps to avoid biasing the results by decreased viability[6365]. PBMCs at 250,000 cells/well were stimulated in duplicate wells with 2 TCID50/PBMC of A/California/7/2009 Pandemic X-179A and A/South Dakota/6/2007 H1N1 influenza viruses, 5 μg/mL phytohemagglutinin (PHA; Sigma) or medium control. After a 48-h incubation at 37°C in a humidified, 5% CO2 atmosphere, plates were washed and stained with anti-IFNγ and GrB mAbs as per manufacturer’s instructions. Spot forming cells (SFCs) were counted with an ImmunoSpot II Analyzer (Cell Technologies Ltd). Results were expressed as SFC/106 PBMC after subtracting the SFC in unstimulated control wells from those enumerated in antigen- or mitogen- stimulated wells.

IgG and IgA FluoroSpot

Cryopreserved PBMCs were used for the detection of IgG and IgA antibody secreting cells (ASC). PBMCs were thawed, counted and then stimulated in RPMI 1640 (Gibco) with 10% fetal bovine serum (Gemini Bio-Products), 0.4% penicillin and streptomycin, 1% Hepes buffer (Corning Cellgrow), 1μg/mL Staphylococcus aureus Cowan (Sigma-Aldrich), 6μg/mL CpG (Operon Technologies), 100ng/mL recombinant human IL-10 (Cell Sciences), and 1μg/mL pokeweed mitogen (Sigma-Aldrich) for 96 hours at 37° C, 5% CO2, which proved to be optimal conditions in our optimization assays. Stimulated PBMCs were used at 50,000 cells/well in duplicate wells of the FluoroSpot IgG/IgA kits (Mabtech Inc.). Assays were performed as per manufacturer’s instructions in plates coated with pH1N1 antigen at 4.1 HA units/well. ASC were enumerated using the ImmunoSpot II instrument. Results were expressed as ASC/106 stimulated PBMCs.

B- and T-cell phenotypic characterization

B- and T-cell subsets were enumerated in freshly thawed cryopreserved PBMCs. After washing and counting viable cells, PBMCs were surface-stained with the following conjugated mAbs: anti-CD3-AF488 (Biolegend; clone HIT3a), anti CD8-APC/Cy7 (Biolegend; SK1), anti-CD19-APC/Cy7 (BD Biosciences; SJ25C1), anti-CD19-PerCP/Cy5.5 (Biolegend; HiB19) anti-CD25-PE/Cy7 (Biolegend; BC96), anti-CD21-PE/Cy7 (BD Biosciences; B-ly4), anti-CD27-PerCP/Cy5.5 (BD Biosciences; M-T271), anti-CD38-PE/Cy7 (Biolegend; HIT2), anti-HLA-DR-PerCP/Cy5.5 (Biolegend; L243); anti-CD10-FITC (BD Biosciences; W8E7), anti-IL21R-PE (BD Biosciences; 17A12), and anti-CD39-APC (Biolegend; A1). Cells were fixed and permeabilized with Cytofix/Cytoperm (BD Biosciences), and stained with anti-IL-10-PE (R & D Systems; 127107), anti-FOXP3-PE (Biolegend; 206D) and anti- TGFβ-PE (Biolegend; TW4-2F8), and analyzed with Guava easyCyte 8HT and FlowJo (Treestar). Subsets were expressed as percentages of the parent CD4+, CD8+ and CD19+ cell populations. The gating strategy is presented in S2 Fig.

Statistical methods

This substudy was an exploratory project and no sample size calculations were performed.* Its objectives included descriptive and correlation analyses, which did not require sample size estimation to generate interpretable results. HAI titers <10 were considered undetectable and were assigned a value of 5. Changes in pH1N1 and sH1N1 HAI titers, T and B-cell mediated immune responses, and phenotypic characterization of T and B-cell subsets from baseline to post immunization or from post-dose 1 to post-dose 2 were assessed using Wilcoxon Matched Pairs Signed-Rank tests. The final flow cytometric analyses were restricted to samples with ≥100 events in the anchor gate. However, a sensitivity analysis showed that the inclusion of samples with <100 events in the anchor gate would not have changed the results. Spearman correlation analyses were performed to assess the strength of associations and test their statistical significance. All analyses were performed using SAS Version 9.2 (SAS Institute INC, Cary, NC) and the graphs were produced using the R software.


Demographic and HIV Disease Characteristics of the Study Population

Of the 119 women on HAART at the time of immunization in the parent study, who received both immunizations prior to delivery and had antibodies measured, 57 had complete sets of PBMCs with adequate viability for the CMI substudy, although in some cases there were not enough cells to perform all CMI assays. The demographic and HIV disease characteristics of the CMI substudy participants did not differ from those of the entire cohort (Table 1). Of the 119 participants, 36 received IIV3 ≥14 days before study entry and 5 received it ≥21 days after the 2nd dose of the pH1N1 IIV1.

pH1N1- and sH1N1-Immune Responses and Their Inter-Relationships


Fig 1 shows HAI antibody titers against pH1N1 and sH1N1 over time in participants who received both doses of pH1N1 vaccine before delivery (consort diagram in S1 Fig). At baseline, the medians of the reciprocal of the HAI titers for pH1N1 and sH1N1 were 10 and 20, respectively. pH1N1 titers significantly increased post-dose 1 of vaccine (median = 80, p<0.001; Fig 1A), but sH1N1 titers did not (p = 0.94; Fig 1B). There were no significant differences in pH1N1 or sH1N1 antibody titers between post-doses 1 and 2 (p = 0.31 and 0.87, respectively).

Fig 1. pH1N1 and sH1N1 HAI titers of HIV-infected pregnant women who received two doses of pH1N1 monovalent vaccine.

Data represent medians and inter quartile ranges of the reciprocals of the HAI titers. The number of subjects who contributed data and p values of paired comparisons are indicated on the graph. Panel A shows pH1N1 antibody titers and panel B shows sH1N1 antibody titers.

Memory B cells (Bmem).

Fig 2 shows the kinetics of pH1N1-specific Bmem IgG and IgA ASC measured by FluoroSpot. There was a significant increase in IgG ASCs post-dose 1 (from median of 6 to 15 ASC/106 PBMC; p = 0.01; Fig 2A), but no further increases post-dose 2 (median = 14 ASC/106 PBMC; p = 0.14) compared to post-dose 1. IgA ASCs were barely detectable at baseline (median = 1 ASC/106 PBMC) and did not significantly increase after vaccination (Fig 2B).

Fig 2. pH1N1 IgG and IgA B-cell memory responses of HIV-infected pregnant women who received two doses of pH1N1 monovalent vaccine.

Data represent medians and inter quartile ranges of antibody secreting cells (ASC)/106 PBMC. The number of subjects who had adequate numbers of viable PBMC to complete the assays at each time point is indicated on the graph. p values calculated with Wilcoxon Matched Pairs Signed-Rank test are indicated on the graph. Panel A shows IgG ASC/106 PBMC and panel B shows IgA ASC/106 PBMC.

Effector T cells (Teff).

pH1N1- and sH1N1-specific Teff were measured by IFNγ and GrB FluoroSpot (Fig 3). Appreciable pH1N1-IFNγ Teff were present at baseline (median = 166 SFC/106 PBMC; Fig 3A) and tended to decrease after vaccination, particularly at post-dose 2 (median = 76 SFC/106 PBMC). In contrast, pH1N1-GrB responses were modest at baseline (median = 48 SFC/106 PBMC; Fig 3B) and did not appreciably change after vaccination. sH1N1 FluoroSpot revealed no significant changes in IFNγ SFCs after either dose of vaccine (Fig 3C), but exhibited a marginally significant increase in GrB SFCs post-dose 1 (p = 0.06; Fig 3D). There were no significant changes in PHA-stimulated IFNγ (Fig 3E) or GrB (Fig 3F) SFCs during the study.

Fig 3. pH1N1 and sH1N1 effector T cell responses of HIV-infected pregnant women who received two doses of pH1N1 monovalent vaccine.

Data represent medians and inter quartile ranges of spot forming cells (SFC)/106 PBMC. The number of subjects who had adequate numbers of viable PBMC to complete the assays at each time point is indicated on the graph. p values calculated with Wilcoxon Matched Pairs Signed-Rank test are indicated on the graph. Panel A shows pH1N1-IFNγ SFC/106 PBMC, panel B pH1N1-granzyme B (GrB) SFC/106 PBMC, panel C sH1N1-IFNγ SFC/106 PBMC, panel D sH1N1-GrB SFC/106 PBMC, panel E PHA-IFNγ SFC/106 PBMC positive controls, and panel F PHA-GrB SFC/106 PBMC controls.

Associations of humoral and cellular pH1N1- and sH1N1-specific responses in pH1N1 IIV1 recipients.

As shown in Table 2, there were significant associations between pH1N1- and sH1N1-HAI titers at all time points. In the subset of women who had Bmem and Teff measured, pH1N1-HAI titers and pH1N1-IFNγ SFCs were significantly correlated at baseline (rho = 0.35; p = 0.02) and post-dose 1 (rho = 0.32; p = 0.04), but only marginally correlated at post-dose 2 (rho = 0.29; p = 0.06). pH1N1-IFNγ and pH1N1-GrB SFCs were highly correlated with each other and with sH1N1-IFNγ and sH1N1-GrB SFCs at all time points (rho≥0.65; p<0.0001). pH1N1 IgG ASCs correlated with pH1N1-IgA ASCs, but not with any other influenza-specific responses.

Table 2. Selected correlations of cellular and humoral B and T cell-mediated immune responses to pH1N1 and sH1N1.

Effect of HIV disease characteristics on immune responses to pH1N1 vaccine

CD4, CD8 and HIV plasma RNA.

Correlation analyses of pH1N1 immune responses with CD4%, CD8% and HIV plasma RNA (Table 3) showed that HAI titers after vaccination significantly increased with higher CD4% and with low CD8%, both post-dose 1 and post-dose 2 (CD4%: rho≥0.32, p≤0.001; CD8%: rho≤-0.24, p≤0.02). There were no significant correlations of HIV plasma RNA with pH1N1-HAI titers after vaccination. In the subset of women who had Bmem and Teff measured, neither IgG nor IgA ASC correlated with CD4%, CD8% or plasma HIV RNA at any time points. However, IFNγ and GrB SFC showed significant negative correlations with HIV plasma RNA at all time points (rho≤-0.37, p≤0.01).

Table 3. Correlations of pH1N1 immune responses with plasma HIV RNA, CD4% and CD8%.

Activated, regulatory, immature and exhausted T and B cell subsets.

To determine whether increased activation or regulation, characteristic of HIV disease, or other HIV-associated abnormalities of lymphocyte subsets played a role in the responses to pH1N1 IIV1, we measured T and B cell subsets representative of activation and regulation and B cell subsets that are typically skewed by HIV infection and may play important roles in antibody responses to vaccines. The following subsets were measured at baseline and after each dose of vaccine: CD19+CD10+, CD19+IL-21R+, CD19+CD27+CD10+ = activated immature B cells, CD19+CD27-CD10+ = immature B cells, CD19+CD27-CD21+ = naïve B cells, CD19+CD27+CD21+ = resting memory B cells, CD19+CD27+CD21- = transitional B cells, CD19+CD27-CD21- = exhausted B cells, CD4+CD39+ = regulatory T cells, CD4+HLADR+CD38+ = activated T cells, CD4+ TGFβ+ = regulatory T cells, CD8+CD39+ = regulatory T cells, CD8+HLADR+CD38+ = activated T cells, CD8+ TGFβ+ = regulatory T cells, CD4+IL10+ = regulatory T cells, CD4+FOXP3+ = regulatory T cells, CD4+CD25+FOXP3+ = regulatory T cells, CD8+IL10+ = regulatory T cells, CD8+FOXP3+ = regulatory T cells, CD8+CD25+FOXP3+ = regulatory T cells, CD19+IL10+ = regulatory B cells and CD19+CD25+ = regulatory B cells. There were no significant changes from baseline to the post-dose 1 and post-dose 2 time points in any of the T or B cell subsets measured (S1 Table).

Correlation analyses of the pH1N1-specific humoral and cellular immune responses with the above listed lymphocyte subsets showed several significant associations (Table 4). HAI antibody responses decreased with high CD8+HLADR+CD38+% activated T cells at all time points (rho≤-0.37, p≤0.02) and marginally increased with CD8+IL10+% regulatory T cells at post-dose 2 (rho = 0.30, p = 0.05). IgG ASC decreased with high CD19+CD27-CD21-% exhausted B cells at all time points (rho≤-0.36, p≤0.03); IgG ASC increased with high CD8+CD39+% regulatory T cells (rho = 0.42, p = 0.02) at post-dose 1 and with high CD19+CD27+CD21-% activated B cells (rho = 0.35, p = 0.04) at post-dose 2. IFNγ SFCs inversely correlated with: CD8+HLADR+CD38+% activated T cells at post-dose 1 (rho = -0.40, p = 0.03) and with CD19+IL10+% regulatory B cells (rho = -0.36, p = 0.047) and marginally with CD4+TGF (rho = -.33, p = 0.07) at post-dose 2.

Table 4. Correlations of pH1N1 immune responses with B- and T-Cell subsets.


This study identified HIV disease characteristics other than CD4 or CD8 cell numbers and HIV viral load that may modulate immune responses to pH1N1 IIV1 in HIV-infected pregnant women. The proportions of activated and regulatory B and T cells and exhausted B cells significantly correlated with pH1N1-specific HAI, Teff, and/or Bmem responses to the vaccine, including some unexpected associations.

The correlation between HAI antibody titers ≥40 and decreased incidence of symptomatic influenza disease suggests that the factors that modulate antibody responses to IIVs are highly relevant for vaccine efficacy. During typical seasonal epidemics, some of the antibody responses are anamnestic, as large proportions of antibodies arise from Bmem[66]. However, during pandemics the primary antibody response is crucial, since adults typically receive a single dose of vaccine to prevent disease. This is especially problematic for HIV-infected patients, who predictably have low antibody responses to influenza vaccines, and for whom there is no recommendation to modify the immunization schedule. In this study, both primary (post-dose 1) and anamnestic (post-dose 2) pH1N1 antibody responses significantly increased with high proportions of CD4 and with low proportions of total CD8 and of CD8 activated T cells, but not with plasma HIV RNA. The proportions of activated T cells in HIV-infected individuals generally mirror viral replication as measured by HIV plasma RNA[67], but in this study, HIV plasma RNA did not correlate with pH1N1-HAI responses. Activated T cells may be elevated even if the plasma viral load is below the limit of detection of conventional HIV RNA PCR assays, and this may be frequently the case shortly after starting cART, which was true for some of the study participants. Other viral and bacterial pathogens, the best known of which is CMV[68], may increase CD8+ activated T cells and may explain the discordance of the effects of CD8+ activated T cells and HIV viral load. A direct mechanism whereby activated T cells suppress de novo immune responses has not been described, however a recent study showed that immune activation decreased immune responses to yellow fever vaccine in individuals without HIV infection[69]. We also previously showed that reconstitution of antigen-specific T-cell responses in HIV-infected children on cART decreased with elevated CD8+ activated T cells[70]. Taken together these data suggest that high frequencies of CD8+ activated T cells may be at least a marker of a dysfunctional immune system and could be used to optimize the timing of vaccine administration to HIV-infected individuals.

Administration of pH1N1 IIV1 generated IgG Bmem after the 1st dose of vaccine without additional increases after the 2nd dose. The magnitude of the pH1N1-Bmem response was similar to that previously described after IIV3 in HIV-infected adults[71,72]. IgA Bmem did not increase after either dose of vaccine, but the magnitude of IgA Bmem significantly correlated with the IgG Bmem at all time points. This is explained by the fact that subjects with high frequencies of IgG Bmem also had relatively high frequencies of IgA Bmem before vaccination and the increase in IgG Bmem after vaccination did not change the rank order of the subject results. The corollary of this observation may be that HIV-infected pregnant women with pre-existing memory to vaccine antigens mount better IgG responses after vaccination than those without pre-existing memory. However, the IgA response is depressed regardless of pre-existing memory. Healthy adults have been previously shown to mount significant IgA Bmem in response to IIV3[71]. The low and heterogeneous IgA Bmem response to IIV1 in our study subjects was likely due to the HIV-associated immune suppression, although an effect of pregnancy cannot be excluded due to the lack of an uninfected control group in this study and the absence of historical information on IgA responses to IIV in pregnant women.

High proportions of exhausted B cells negatively impacted the magnitude of the pH1N1-IgG Bmem responses to vaccination. HIV infection is characterized by an increase in activated B cells, exhausted B cells and immature B-cell subsets. The effect of the change in B-cell subset relative proportions on vaccine responses during pregnancy complicated by HIV infection has not been previously characterized. Although increased proportions of exhausted B cells attenuated the Bmem responses to pH1N1 vaccine, the increased proportions of immature B cells did not affect the IgG Bmem responses. High proportions of activated B cells after the 2nd dose of vaccine were significantly associated with increased pH1N1-IgG Bmem responses, suggesting that vigorous anamnestic Bmem responses to vaccines increase the frequency of circulating activated B cells. cART decreases polyclonal activated B cells and partially corrects the distribution of other B-cell subsets; however, in HIV-infected subjects, even after sustained inhibition of the HIV replication, the resting Bmem subset continues to be lower and the activated, exhausted and immature B cell subsets remain higher compared with HIV-uninfected age-matched controls[54,60] offering a potential explanation for low vaccine immunogenicity in HIV-infected individuals on cART. Although early initiation of cART has a better restorative effect on the B-cell compartment compared with initiation in the chronic phase of HIV infection, recent reports showed a lack of normalization of the B cell subsets even in early-treated individuals[73]. Taken together, these observations suggest that defective Bmem responses to IIV, and perhaps to other inactivated vaccines, may continue as a sustained complication of HIV infection, and that persistence of vaccine-conferred antibody-mediated protection may be different in HIV-infected compared with uninfected individuals. This underscores the importance of studying the persistence of antibodies and the ability to elicit anamnestic responses to vaccines in HIV-infected individuals.

pH1N1-Bmem paradoxically increased after the 1st dose of vaccine with high CD8+CD39+% Tregs. CD39 is an ectonucleosidase that hydrolyzes ATP and ADP[74], and in conjunction with CD73, which is usually co-expressed with CD39, hydrolyzes extracellular ATP to adenosine. This results in a reduction of the pro-inflammatory activity of extracellular ATP and an increase of the anti-inflammatory effect of adenosine. This mechanism is deemed to mediate the regulatory activity of CD39+ regulatory T cells. However, extracellular adenosine also contributes to immunoglobulin class switch[75], which may explain the association found in this study between the frequency of CD8+CD39+ regulatory T cells and pH1N1-IgG Bmem responses to primary immunization. Alternatively, the relationship between IgG Bmem and CD8+CD39+% Treg might not be causational.

The generation of influenza-specific Teff in response to IIV1 is also important because it provides a second line of protection against severe infection. If antibody-mediated protection is insufficient to prevent infection, the Teff become responsible for eliminating influenza-infected cells and limiting viral spread. The second dose of pH1N1 IIV1 resulted in a decrease of pH1N1-specific IFNγ Teff. Although pH1N1-GrB Teff did not change after vaccination, they were highly associated with IFNγ Teff at all time points. The low and heterogeneous GrB Teff responses may reflect the low CD8+ T cell stimulatory ability of IIVs. pH1N1-Teff significantly decreased with high plasma HIV load, activated CD8+ T cells and regulatory B cells and moderately decreased with CD8+CD39+, CD4+TGFβ+ and CD4+IL10+ regulatory T cells. Both HIV infection and pregnancy increase the proportions of regulatory T cells[7678], but regulatory T cells associated with HIV infection probably play the dominant role in attenuating Teff responses to IIV, since the decrease of Teff after influenza vaccination in previously primed non-pregnant HIV-infected individuals has been a recurrent and consistent observation in our studies [79,80]. CMI decreases were not observed in HIV-uninfected individuals after IIV3[81]. In a previous study, we showed that pH1N1-specific CD8+FOXP3+ and CD8+TGFβ+ regulatory T cells increased after the first dose of pH1N1 IIV1 in HIV-infected children and youth[80]. Although these are not the same regulatory T cell subsets that were found to be associated with decreased pH1N1-IFNγ Teff in this study, this observation suggests that pH1N1 IIV1 administration may lead to an increase in regulatory T cells that may dampen subsequent recall Teff responses. Further studies are needed to determine the fine specificity of circulating regulatory T cells after IIV administration and their ability to block the expansion of influenza-specific Teff.

It is important to note the association of pH1N1 and sH1N1 antibody and Teff responses at all time points. pH1N1 and the immediately preceding sH1N1 shared approximately 70% of the T-cell epitopes[82], such that cross reactivity is the most likely explanation of the strong Teff correlations. In contrast, there was practically no B-cell epitope homology between pH1N1 and sH1N1, which makes Bmem cross reactivity unlikely. A potential explanation of the positive correlation between sH1N1 and pH1N1 antibody titers is that in our study participants the magnitude of the antibody responses to sH1N1 vaccination or infection was likely limited by the same factors that limited the magnitude of the antibody responses to pH1N1 IIV1. Alternatively, sH1N1 and pH1N1 cross-reactive helper T cells may have contributed to the increased antibody responses to pH1N1 in subjects with pre-existing immunity to sH1N1. This latter hypothesis is also in agreement with the association between pH1N1 HAI titers and IFNγ SFC observed in our study.

pH1N1-HAI titers significantly correlated with IFNγ SFC at baseline and post-dose 1, but only marginally at post-dose 2. The baseline and post-dose 1 correlations are in agreement with the Th1-dependence of influenza antibody responses [83,84]. In addition, at entry, 21% of the study population had serologic evidence of past wild-type pH1N1 infection, which probably contributed to the baseline correlation between pH1N1-HAI and Teff, since presumably participants with past infection had higher HAI titers and IFNγ SFC compared with those without past infection. The lack of correlation between pH1N1-Bmem and pH1N1-HAI titers was surprising and contrasted with our previous findings in HIV-infected children and non-pregnant youth. Although the limited number of data points available for these analyses may have also limited the ability to detect the correlations above, this observation needs to be further explored in the context of pregnancy.

This study had several limitations. The use of a single regimen, which included 2 double-doses of pH1N1 IIV1, did not allow precise determination of the effect of increasing the amount of antigen and of the number of doses on the immune response to the vaccine. Studies comparing the immunogenicity of double and standard doses of pH1N1 IIV1 in other HIV-infected populations reported contradictory results[8587]. The second dose of vaccine in our study did not significantly improve pH1N1-antibody or CMI, which is in agreement with other reports[85]. Our results also have to be interpreted with caution due to the lack of information on correlates of protection against influenza infection and/or disease in HIV-infected individuals.

For the two measures of IIV immunogenicity that are mechanistically associated with protection against influenza infection and disease, antibodies and Teff, our study showed a significant handicap in HIV-infected pregnant women compared with uninfected, nonpregnant historical controls: antibody responses were lower than those of historical controls and IFNγ Teff decreased after multiple doses of vaccine. The decreased immunogenicity of pH1N1 IIV1 was most closely correlated with increased HIV replication and with high proportions of activated and regulatory T cells and exhausted and regulatory B cells. Pregnancy, which is associated with increased frequencies of regulatory T cells, may contribute to the decreased immunogenicity of the vaccine as suggested by recent studies[18,19]. The effects of HIV treatment on the parameters that were associated with low immunogenicity of pH1N1 in this study appear to be as follows: HIV replication can be controlled with appropriate cART, but there is a dearth of information on regulatory B cells in the context of treated or untreated HIV infection and no evidence yet that activated and regulatory T cells and exhausted B cells completely normalize with cART. Until such evidence is presented, special immunization regimens or vaccines with enhanced immunogenicity (e.g., adjuvanted vaccines) need to be considered for optimizing the protection conferred by IIV to HIV-infected pregnant women.

Supporting Information

S2 Fig. Gating strategy.

The figure illustrates a typical example of the gating strategy. Lymphocyte were identified by forward and side scatter. Next, CD4+ T cells were identified as being CD3+CD8- lymphocytes and CD8+ T cells as CD3+CD8+ lymphocytes. Next, the subset of interest was identified by the expression of its characteristic marker. Examples are shown for CD4+TGFb+, CD4+CD39+, CD4+HLADR+CD38+ subsets and their CD8+ counterparts. B cells were identified as CD19+CD3- lymphocytes (not shown).


S1 Table. Phenotypic Characterization of B- and T-Cell Subsets at Baseline and after pH1N1 Vaccination.



The authors appreciate the contributions of the women and infants who participated in this study and the assistance of research personnel at the study sites.

Additional members of the P1086 Protocol Team

George Siberry, MD, MPH, Pediatric Adolescent and Maternal AIDS Branch, Eunice Kennedy Shriver National Institute of Child Health and Human Development, Bethesda, Maryland; Judi Miller, RN, BSN, Division of AIDS, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland; Elizabeth Petzold, PhD, Social & Scientific Systems, Silver Spring, Maryland; Wende Levy, RN, MS, Social & Scientific Systems, Silver Spring, Maryland; Barbara Heckman, BS, Frontier Science and Technology Research Foundation, Buffalo, New York; Ruth Ebiasah, PharmD, MS, RPh, Division of AIDS, National Institute of Allergy and Infectious Diseases, Bethesda, MD; Paul Palumbo, MD, Dartmouth-Hitchcock Medical Center, Lebanon, New Hampshire; Joan Dragavon, MLM, University of Washington, Seattle, Washington; Lori Donelson, RN, BSN, CCRC, Westat, Rockville, Maryland; Andrea Jurgrau, MSRN, CPNP, Columbia-Presbyterian Medical Center, New York, New York; David Garry, DO, Jacobi Medical Center, Bronx, New York.

Participating sites and site personnel

University of Miami Pediatric/Perinatal HIV/AIDS CRS (Amanda Cotter, MD; Gwendolyn B. Scott, MD; Erika Lopez-Bertiery, MD; Safia Khan, MD); University of Puerto Rico Pediatric HIV/AIDS Research Program CRS (Irma L. Febo, MD; Carmen D. Zorrilla, MD; Vivian Tamayo-Agrait, MD; Ruth Santos, RN, MPH); University of Southern California, LA NICHD CRS (Alice Stek, MD; Michael Neely, MD; LaShonda Spencer, MD; Andrea Kovacs, MD);Washington Hospital Center NICHD CRS (Sara Parker, MD; Patricia Tanjutco, MD; Vanessa Emmanuel, BA; Liv Thulin); Texas Children’s Hospital CRS (Shelly Buschur, RN, CNM; Mary Paul, MD; Filiz Seeborg, MD; Kathy Pitts, PhD); Chicago Children's CRS (Jessica Shore, RN; Sarah Sutton, MD); UCSD Maternal, Child, and Adolescent HIV CRS (Stephen A. Spector, MD; Andrew Hull, MD; Mary Caffery, RN, MSN; Jean Manning, RN, BSN); DUMC Pediatric CRS (Margaret Donnelly, PA; Mary Jo Hassett, RN; Elizabeth Livingston, MD; Julieta Giner, RN); Rush University Cook County Hospital, Chicago NICHD CRS (Mariam Aziz, MD; Latania Logan, MD; Julie Schmidt, MD; Helen Cejtin, MD); The Children's Hospital of Philadelphia IMPAACT CRS (Samuel Parry, MD; Rita Leite, MD); University of South Florida, Tampa NICHD CRS (Karen L. Bruder, MD; Gail Lewis, RN; Patricia Emmanuel, MD; Tampa General Hospital); San Juan City Hospital PR NICHD CRS (Elvia Perez, MPH, MA; Rodrigo Diaz, MD; Dalila Guzman, RPh; Midnela Acevedo-Flores, MD); South Florida CDC, Ft Lauderdale NICHD CRS; Johns Hopkins University, Baltimore NCHD CRS (Allison Agwu, MD, ScM; Todd Noletto, MPH; Jennifer Chang, BS; Andi Weiss, PharmD); Tulane University, New Orleans NICHD CRS (Chi Dola, MD; Thomas Alchediak, MD; Yvette Luster, RN; Sheila Bradford, RN); Bronx-Lebanon Hospital IMPAACT CRS (Jenny Gutierrez, MD; Mahboobullah Mirza Baig, MD; Stefan Hagmann, MD; Murli Purswani, MD); NJ Medical School CRS (Arlene D. Bardeguez, MD, MPH; Charmane Calilap-Bernardo, RN; Linda Bettica, RN); Columbia IMPAACT CRS (Andrea Jurgrau, CNP; Gina Silva, RN; Alice Higgins, RN; Marc Foca, MD); Metropolitan Hospital NICHD CRS (Mahrukh Bamji, MD; Santa Paul, MD; Siobhan Riley, MPH; Deepali Jain, MD); Children's Hospital of Boston NICHD CRS (Sandra K. Burchett, MD, MS; Ruth Tuomala, MD; Arlene Buck, RN; Catherine Kneut, RN, CPNP); University of Colorado, Denver NICHD CRS (Jennifer Dunn, FNP-C; Paul Harding, MS; Kay Kinzie, FNP-C; Jenna Wallace, MSW; Supported by NIH/NCATS Colorado CTSI Grant Number UL1 TR000154); St. Jude/UTHSC CRS (L. Jill Utech, RN, MSN, CCRP; Edwin Thorpe, Jr, MD; Nina Sublette, RN, FNP, PhD; Pam Finnie, MSN); WNE Maternal Pediatric Adolescent AIDS CRS; UCLA-Los Angeles/Brazil AIDS Consortium (LABAC) CRS (Jaime G. Deville, MD; Karin Nielsen-Saines, MD; Nicole Falgout, RN; Joseph Geffen); Boston Medical Center Pediatric HIV Program NICHD CRS; Jacobi Medical Center Bronx NICHD CRS; Seattle Children's Hospital CRS; SUNY Stony Brook NICHD CRS (Denise Ferraro, FNP; Erin Infanzon; Michele Kelly, NP; Jennifer Griffin, CNM); Miller Children's Hospital Long Beach, CA NICHD CRS (Audra Deveikis, MD; Janielle Jackson-Alvarez, RN; Tempe K. Chen, MD; Jagmohan S. Batra, MD); University of Florida College of Medicine, Jacksonville NICHD CRS (Mobeen Rathore, MD; Ayesha Mirza, MD; Nizar Maraqa, MD; Kathleen Thoma, MA, CCRP); University of California, San Francisco NICHD CRS (Diane Wara, MD; Nicole Tilton, PNP; Mica Muscat, PNP.

Overall support for the International Maternal Pediatric Adolescent AIDS Clinical Trials Group (IMPAACT) was provided by the National Institute of Allergy and Infectious Diseases (NIAID) [U01 AI068632], the Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD), and the National Institute of Mental Health (NIMH) [AI068632]. Support of the sites was provided by the National Institute of Allergy and Infectious Diseases (NIAID) and the NICHD International and Domestic Pediatric and Maternal HIV Clinical Trials Network funded by NICHD (contract number N01-DK-9-001/HHSN267200800001C). The laboratory work was supported by N01HD33162 (97–07) and the statistical work by the Statistical and Data Analysis Center at Harvard School of Public Health, under the National Institute of Allergy and Infectious Diseases cooperative agreement #5 U01 AI41110 with the Pediatric AIDS Clinical Trials Group (PACTG) and #1 U01 AI068616 with the IMPAACT Group. The contents of this publication are solely the responsibility of the authors and do not necessarily represent the official views of the NIH, the United States Government, the U.S. Department of State.

Author Contributions

Conceived and designed the experiments: AW. Performed the experiments: KMR TD. Analyzed the data: AW PM KMR TD TF. Contributed reagents/materials/analysis tools: AW PM KMR TD TF. Wrote the paper: AW PM KMR TF TD AB DHW MJA SAN MJL.


  1. 1. Dolan GP, Myles PR, Brett SJ, Enstone JE, Read RC, Openshaw PJ, et al. The comparative clinical course of pregnant and non-pregnant women hospitalised with influenza A(H1N1)pdm09 infection. PloS one. 2012;7:e41638. pmid:22870239
  2. 2. Dubar G, Azria E, Tesniere A, Dupont H, Le Ray C, Baugnon T, et al. French experience of 2009 A/H1N1v influenza in pregnant women. PloS one. 2010;5.
  3. 3. Hewagama S, Walker SP, Stuart RL, Gordon C, Johnson PD, Friedman ND, et al. 2009 H1N1 influenza A and pregnancy outcomes in Victoria, Australia. Clinical infectious diseases: an official publication of the Infectious Diseases Society of America. 2010;50:686–90. pmid:20100064
  4. 4. Louie JK, Acosta M, Jamieson DJ, Honein MA. Severe 2009 H1N1 influenza in pregnant and postpartum women in California. The New England journal of medicine. 2010;362:27–35. pmid:20032319
  5. 5. Siston AM, Rasmussen SA, Honein MA, Fry AM, Seib K, Callaghan WM, et al. Pandemic 2009 influenza A(H1N1) virus illness among pregnant women in the United States. JAMA: the journal of the American Medical Association. 2010;303:1517–25. pmid:20407061
  6. 6. Neuzil KM, Reed GW, Mitchel EF, Simonsen L, Griffin MR. Impact of influenza on acute cardiopulmonary hospitalizations in pregnant women. American journal of epidemiology. 1998;148:1094–102. pmid:9850132
  7. 7. Cox S, Posner SF, McPheeters M, Jamieson DJ, Kourtis AP, Meikle S. Hospitalizations with respiratory illness among pregnant women during influenza season. Obstetrics and gynecology. 2006;107:1315–22. pmid:16738158
  8. 8. Stanwell-Smith R, Parker AM, Chakraverty P, Soltanpoor N, Simpson CN. Possible association of influenza A with fetal loss: investigation of a cluster of spontaneous abortions and stillbirths. Communicable disease report CDR review. 1994;4:R28–32. pmid:7513232
  9. 9. Fukushima W, Ohfuji S, Deguchi M, Kawabata K, Hatayama H, Yoshida H, et al. Effectiveness of an influenza A (H1N1) 2009 monovalent vaccine among Japanese pregnant women: a prospective observational study assessing antibody efficacy. Vaccine. 2012;30:7630–6. pmid:23085364
  10. 10. Zaman K, Roy E, Arifeen SE, Rahman M, Raqib R, Wilson E, et al. Effectiveness of maternal influenza immunization in mothers and infants. The New England journal of medicine. 2008;359:1555–64. pmid:18799552
  11. 11. Adedinsewo DA, Noory L, Bednarczyk RA, Steinhoff MC, Davis R, Ogbuanu C, et al. Impact of maternal characteristics on the effect of maternal influenza vaccination on fetal outcomes. Vaccine. 2013;31:5827–33. pmid:24120677
  12. 12. Benowitz I, Esposito DB, Gracey KD, Shapiro ED, Vazquez M. Influenza vaccine given to pregnant women reduces hospitalization due to influenza in their infants. Clinical infectious diseases: an official publication of the Infectious Diseases Society of America. 2010;51:1355–61. pmid:21058908
  13. 13. Eick AA, Uyeki TM, Klimov A, Hall H, Reid R, Santosham M, et al. Maternal influenza vaccination and effect on influenza virus infection in young infants. Archives of pediatrics & adolescent medicine. 2011;165:104–11.
  14. 14. Maltezou HC, Fotiou A, Antonakopoulos N, Kallogriopoulou C, Katerelos P, Dimopoulou A, et al. Impact of postpartum influenza vaccination of mothers and household contacts in preventing febrile episodes, influenza-like illness, healthcare seeking, and administration of antibiotics in young infants during the 2012–2013 influenza season. Clinical infectious diseases: an official publication of the Infectious Diseases Society of America. 2013;57:1520–6.
  15. 15. Omer SB, Goodman D, Steinhoff MC, Rochat R, Klugman KP, Stoll BJ, et al. Maternal influenza immunization and reduced likelihood of prematurity and small for gestational age births: a retrospective cohort study. PLoS medicine. 2011;8:e1000441. pmid:21655318
  16. 16. Omer SB, Zaman K, Roy E, Arifeen SE, Raqib R, Noory L, et al. Combined effects of antenatal receipt of influenza vaccine by mothers and pneumococcal conjugate vaccine receipt by infants: results from a randomized, blinded, controlled trial. The Journal of infectious diseases. 2013;207:1144–7. pmid:23300160
  17. 17. Englund JA, Mbawuike IN, Hammill H, Holleman MC, Baxter BD, Glezen WP. Maternal immunization with influenza or tetanus toxoid vaccine for passive antibody protection in young infants. The Journal of infectious diseases. 1993;168:647–56. pmid:8354906
  18. 18. Bischoff AL, Folsgaard NV, Carson CG, Stokholm J, Pedersen L, Holmberg M, et al. Altered response to A(H1N1)pnd09 vaccination in pregnant women: a single blinded randomized controlled trial. PloS one. 2013;8:e56700. pmid:23637733
  19. 19. Schlaudecker EP, McNeal MM, Dodd CN, Ranz JB, Steinhoff MC. Pregnancy modifies the antibody response to trivalent influenza immunization. The Journal of infectious diseases. 2012;206:1670–3. pmid:22984116
  20. 20. Cohen C, Simonsen L, Sample J, Kang JW, Miller M, Madhi SA, et al. Influenza-related mortality among adults aged 25–54 years with AIDS in South Africa and the United States of America. Clinical infectious diseases: an official publication of the Infectious Diseases Society of America. 2012;55:996–1003.
  21. 21. Fine AD, Bridges CB, De Guzman AM, Glover L, Zeller B, Wong SJ, et al. Influenza A among patients with human immunodeficiency virus: an outbreak of infection at a residential facility in New York City. Clinical infectious diseases: an official publication of the Infectious Diseases Society of America. 2001;32:1784–91.
  22. 22. Klein MB, Lu Y, DelBalso L, Cote S, Boivin G. Influenzavirus infection is a primary cause of febrile respiratory illness in HIV-infected adults, despite vaccination. Clinical infectious diseases: an official publication of the Infectious Diseases Society of America. 2007;45:234–40.
  23. 23. Madhi SA, Kuwanda L, Venter M, Violari A. Prospective cohort study comparing seasonal and H1N1(2009) pandemic influenza virus illnesses in HIV-infected children during 2009. The Pediatric infectious disease journal. 2014;33:174–6. pmid:23907261
  24. 24. Martinez E, Marcos MA, Hoyo-Ulloa I, Anton A, Sanchez M, Vilella A, et al. Influenza A H1N1 in HIV-infected adults. HIV medicine. 2011;12:236–45. pmid:21255221
  25. 25. Noguera-Julian A, Provens AC, Soler-Palacin P, Espiau M, Mur A, Mendez M, et al. Pandemic influenza a (2009 H1N1) in human immunodeficiency virus-infected catalan children. The Pediatric infectious disease journal. 2011;30:173–5. pmid:20802374
  26. 26. Peters PJ, Skarbinski J, Louie JK, Jain S, Roland M, Jani SG, et al. HIV-infected hospitalized patients with 2009 pandemic influenza A (pH1N1)—United States, spring and summer 2009. Clinical infectious diseases: an official publication of the Infectious Diseases Society of America. 2011;52 Suppl 1:S183–8.
  27. 27. Richardson K, Weinberg A. Reduced immunogenicity of influenza vaccines in HIV-infected compared with uninfected pregnant women is associated with regulatory T cells. AIDS. 2011;25:595–602. pmid:21368590
  28. 28. Chadwick EG, Chang G, Decker MD, Yogev R, Dimichele D, Edwards KM. Serologic response to standard inactivated influenza vaccine in human immunodeficiency virus-infected children. The Pediatric infectious disease journal. 1994;13:206–11. pmid:8177629
  29. 29. Kroon FP, van Dissel JT, de Jong JC, van Furth R. Antibody response to influenza, tetanus and pneumococcal vaccines in HIV-seropositive individuals in relation to the number of CD4+ lymphocytes. AIDS. 1994;8:469–76. pmid:7912086
  30. 30. Miotti PG, Nelson KE, Dallabetta GA, Farzadegan H, Margolick J, Clements ML. The influence of HIV infection on antibody responses to a two-dose regimen of influenza vaccine. JAMA: the journal of the American Medical Association. 1989;262:779–83.
  31. 31. Belshe RB, Gruber WC, Mendelman PM, Mehta HB, Mahmood K, Reisinger K, et al. Correlates of immune protection induced by live, attenuated, cold-adapted, trivalent, intranasal influenza virus vaccine. J Infect Dis. 2000;181:1133–7. pmid:10720541
  32. 32. Levin MJ, Song LY, Fenton T, Nachman S, Patterson J, Walker R, et al. Shedding of live vaccine virus, comparative safety, and influenza-specific antibody responses after administration of live attenuated and inactivated trivalent influenza vaccines to HIV-infected children. Vaccine. 2008;26:4210–7. pmid:18597900
  33. 33. Vigano A, Zuccotti GV, Pacei M, Erba P, Castelletti E, Giacomet V, et al. Humoral and cellular response to influenza vaccine in HIV-infected children with full viroimmunologic response to antiretroviral therapy. J Acquir Immune Defic Syndr. 2008;48:289–96. pmid:18545155
  34. 34. Benne CA, Kroon FP, Harmsen M, Tavares L, Kraaijeveld CA, De Jong JC. Comparison of neutralizing and hemagglutination-inhibiting antibody responses to influenza A virus vaccination of human immunodeficiency virus-infected individuals. Clinical and diagnostic laboratory immunology. 1998;5:114–7. pmid:9455891
  35. 35. Flynn PM, Nachman S, Muresan P, Fenton T, Spector SA, Cunningham CK, et al. Safety and immunogenicity of 2009 pandemic H1N1 influenza vaccination in perinatally HIV-1-infected children, adolescents, and young adults. The Journal of infectious diseases. 2012;206:421–30. pmid:22615311
  36. 36. 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. Clinical infectious diseases: an official publication of the Infectious Diseases Society of America. 1999;28:541–7.
  37. 37. Weinberg A, Song LY, Walker R, Allende M, Fenton T, Patterson-Bartlett J, et al. Anti-influenza serum and mucosal antibody responses after administration of live attenuated or inactivated influenza vaccines to HIV-infected children. Journal of acquired immune deficiency syndromes. 2010;55:189–96. pmid:20581690
  38. 38. Hobson D, Curry RL, Beare AS, Ward-Gardner A. The role of serum haemagglutination-inhibiting antibody in protection against challenge infection with influenza A2 and B viruses. The Journal of hygiene. 1972;70:767–77. pmid:4509641
  39. 39. Black S, Nicolay U, Vesikari T, Knuf M, Del Giudice G, Della Cioppa G, et al. Hemagglutination inhibition antibody titers as a correlate of protection for inactivated influenza vaccines in children. The Pediatric infectious disease journal. 2011;30:1081–5. pmid:21983214
  40. 40. Pedersen GK, Madhun AS, Breakwell L, Hoschler K, Sjursen H, Pathirana RD, et al. T-helper 1 cells elicited by H5N1 vaccination predict seroprotection. The Journal of infectious diseases. 2012;206:158–66. pmid:22551811
  41. 41. Eto D, Lao C, DiToro D, Barnett B, Escobar TC, Kageyama R, et al. IL-21 and IL-6 are critical for different aspects of B cell immunity and redundantly induce optimal follicular helper CD4 T cell (Tfh) differentiation. PloS one. 2011;6:e17739. pmid:21423809
  42. 42. Cubas RA, Mudd JC, Savoye AL, Perreau M, van Grevenynghe J, Metcalf T, et al. Inadequate T follicular cell help impairs B cell immunity during HIV infection. Nature medicine. 2013;19:494–9. pmid:23475201
  43. 43. Pallikkuth S, Parmigiani A, Silva SY, George VK, Fischl M, Pahwa R, et al. Impaired peripheral blood T-follicular helper cell function in HIV-infected nonresponders to the 2009 H1N1/09 vaccine. Blood. 2012;120:985–93. pmid:22692510
  44. 44. Pallikkuth S, Pilakka Kanthikeel S, Silva SY, Fischl M, Pahwa R, Pahwa S. Upregulation of IL-21 receptor on B cells and IL-21 secretion distinguishes novel 2009 H1N1 vaccine responders from nonresponders among HIV-infected persons on combination antiretroviral therapy. Journal of immunology. 2011;186:6173–81. pmid:21531891
  45. 45. Moir S, Fauci AS. B cells in HIV infection and disease. Nature reviews Immunology. 2009;9:235–45. pmid:19319142
  46. 46. Imbeault M, Ouellet M, Giguere K, Bertin J, Belanger D, Martin G, et al. Acquisition of host-derived CD40L by HIV-1 in vivo and its functional consequences in the B-cell compartment. Journal of virology. 2011;85:2189–200. pmid:21177803
  47. 47. Martin G, Roy J, Barat C, Ouellet M, Gilbert C, Tremblay MJ. Human immunodeficiency virus type 1-associated CD40 ligand transactivates B lymphocytes and promotes infection of CD4+ T cells. Journal of virology. 2007;81:5872–81. pmid:17392362
  48. 48. Moir S, Malaspina A, Pickeral OK, Donoghue ET, Vasquez J, Miller NJ, et al. Decreased survival of B cells of HIV-viremic patients mediated by altered expression of receptors of the TNF superfamily. The Journal of experimental medicine. 2004;200:587–99. pmid:15508184
  49. 49. Perise-Barrios AJ, Munoz-Fernandez MA, Pion M. Direct phenotypical and functional dysregulation of primary human B cells by human immunodeficiency virus (HIV) type 1 in vitro. PloS one. 2012;7:e39472. pmid:22768302
  50. 50. Qiao X, He B, Chiu A, Knowles DM, Chadburn A, Cerutti A. Human immunodeficiency virus 1 Nef suppresses CD40-dependent immunoglobulin class switching in bystander B cells. Nature immunology. 2006;7:302–10. pmid:16429138
  51. 51. Viau M, Veas F, Zouali M. Direct impact of inactivated HIV-1 virions on B lymphocyte subsets. Molecular immunology. 2007;44:2124–34. pmid:17134757
  52. 52. Rappocciolo G, Piazza P, Fuller CL, Reinhart TA, Watkins SC, Rowe DT, et al. DC-SIGN on B lymphocytes is required for transmission of HIV-1 to T lymphocytes. PLoS pathogens. 2006;2:e70. pmid:16839201
  53. 53. Moir S, Ho J, Malaspina A, Wang W, DiPoto AC, O'Shea MA, et al. Evidence for HIV-associated B cell exhaustion in a dysfunctional memory B cell compartment in HIV-infected viremic individuals. The Journal of experimental medicine. 2008;205:1797–805. pmid:18625747
  54. 54. Pensieroso S, Galli L, Nozza S, Ruffin N, Castagna A, Tambussi G, et al. B-cell subset alterations and correlated factors in HIV-1 infection. AIDS. 2013;27:1209–17. pmid:23343911
  55. 55. Titanji K, De Milito A, Cagigi A, Thorstensson R, Grutzmeier S, Atlas A, et al. Loss of memory B cells impairs maintenance of long-term serologic memory during HIV-1 infection. Blood. 2006;108:1580–7. pmid:16645169
  56. 56. Fontaine J, Chagnon-Choquet J, Valcke HS, Poudrier J, Roger M. High expression levels of B lymphocyte stimulator (BLyS) by dendritic cells correlate with HIV-related B-cell disease progression in humans. Blood. 2011;117:145–55. pmid:20870901
  57. 57. Hart M, Steel A, Clark SA, Moyle G, Nelson M, Henderson DC, et al. Loss of discrete memory B cell subsets is associated with impaired immunization responses in HIV-1 infection and may be a risk factor for invasive pneumococcal disease. Journal of immunology. 2007;178:8212–20. pmid:17548660
  58. 58. Ho J, Moir S, Malaspina A, Howell ML, Wang W, DiPoto AC, et al. Two overrepresented B cell populations in HIV-infected individuals undergo apoptosis by different mechanisms. Proceedings of the National Academy of Sciences of the United States of America. 2006;103:19436–41. pmid:17158796
  59. 59. Swingler S, Brichacek B, Jacque JM, Ulich C, Zhou J, Stevenson M. HIV-1 Nef intersects the macrophage CD40L signalling pathway to promote resting-cell infection. Nature. 2003;424:213–9. pmid:12853962
  60. 60. Jacobsen MC, Thiebaut R, Fisher C, Sefe D, Clapson M, Klein N, et al. Pediatric human immunodeficiency virus infection and circulating IgD+ memory B cells. The Journal of infectious diseases. 2008;198:481–5. pmid:18582200
  61. 61. Levesque MC, Moody MA, Hwang KK, Marshall DJ, Whitesides JF, Amos JD, et al. Polyclonal B cell differentiation and loss of gastrointestinal tract germinal centers in the earliest stages of HIV-1 infection. PLoS medicine. 2009;6:e1000107. pmid:19582166
  62. 62. Abzug MJ, Nachman SA, Muresan P, Handelsman E, Watts DH, Fenton T, et al. Safety and immunogenicity of 2009 pH1N1 vaccination in HIV-infected pregnant women. Clinical infectious diseases: an official publication of the Infectious Diseases Society of America. 2013;56:1488–97.
  63. 63. Weinberg A, Song LY, Wilkening CL, Fenton T, Hural J, Louzao R, et al. Optimization of storage and shipment of cryopreserved peripheral blood mononuclear cells from HIV-infected and uninfected individuals for ELISPOT assays. J Immunol Methods. 2010;363:42–50. pmid:20888337
  64. 64. Weinberg A, Song LY, Wilkening C, Sevin A, Blais B, Louzao R, et al. Optimization and limitations of use of cryopreserved peripheral blood mononuclear cells for functional and phenotypic T-cell characterization. Clin Vaccine Immunol. 2009;16:1176–86. pmid:19515870
  65. 65. Bull M, Lee D, Stucky J, Chiu YL, Rubin A, Horton H, et al. Defining blood processing parameters for optimal detection of cryopreserved antigen-specific responses for HIV vaccine trials. J Immunol Methods. 2007;322:57–69. pmid:17382342
  66. 66. Bentebibel SE, Lopez S, Obermoser G, Schmitt N, Mueller C, Harrod C, et al. Induction of ICOS+CXCR3+CXCR5+ TH cells correlates with antibody responses to influenza vaccination. Science translational medicine. 2013;5:176ra32. pmid:23486778
  67. 67. Hatano H, Jain V, Hunt PW, Lee TH, Sinclair E, Do TD, et al. Cell-based measures of viral persistence are associated with immune activation and programmed cell death protein 1 (PD-1)-expressing CD4+ T cells. The Journal of infectious diseases. 2013;208:50–6. pmid:23089590
  68. 68. Appay V, Sauce D. Immune activation and inflammation in HIV-1 infection: causes and consequences. The Journal of pathology. 2008;214:231–41. pmid:18161758
  69. 69. Muyanja E, Ssemaganda A, Ngauv P, Cubas R, Perrin H, Srinivasan D, et al. Immune activation alters cellular and humoral responses to yellow fever 17D vaccine. The Journal of clinical investigation. 2014;124:1. pmid:24642469
  70. 70. Weinberg A, Pahwa S, Oyomopito R, Carey VJ, Zimmer B, Mofenson L, et al. Antimicrobial-specific cell-mediated immune reconstitution in children with advanced human immunodeficiency virus infection receiving highly active antiretroviral therapy. Clinical infectious diseases: an official publication of the Infectious Diseases Society of America. 2004;39:107–14. pmid:15206061
  71. 71. Sasaki S, Jaimes MC, Holmes TH, Dekker CL, Mahmood K, Kemble GW, et al. Comparison of the influenza virus-specific effector and memory B-cell responses to immunization of children and adults with live attenuated or inactivated influenza virus vaccines. Journal of virology. 2007;81:215–28. pmid:17050593
  72. 72. Ho J, Moir S, Wang W, Posada JG, Gu W, Rehman MT, et al. Enhancing effects of adjuvanted 2009 pandemic H1N1 influenza A vaccine on memory B-cell responses in HIV-infected individuals. AIDS. 2011;25:295–302. pmid:21157297
  73. 73. Moir S, Fauci AS. Insights into B cells and HIV-specific B-cell responses in HIV-infected individuals. Immunological reviews. 2013;254:207–24. pmid:23772622
  74. 74. Antonioli L, Pacher P, Vizi ES, Hasko G. CD39 and CD73 in immunity and inflammation. Trends in molecular medicine. 2013;19:355–67. pmid:23601906
  75. 75. Schena F, Volpi S, Faliti CE, Penco F, Santi S, Proietti M, et al. Dependence of immunoglobulin class switch recombination in B cells on vesicular release of ATP and CD73 ectonucleotidase activity. Cell reports. 2013;3:1824–31. pmid:23770243
  76. 76. Chougnet CA, Shearer GM. Regulatory T cells (Treg) and HIV/AIDS: summary of the September 7–8, 2006 workshop. AIDS Res Hum Retroviruses. 2007;23:945–52. pmid:17678480
  77. 77. Zenclussen AC, Gerlof K, Zenclussen ML, Sollwedel A, Bertoja AZ, Ritter T, et al. Abnormal T-cell reactivity against paternal antigens in spontaneous abortion: adoptive transfer of pregnancy-induced CD4+CD25+ T regulatory cells prevents fetal rejection in a murine abortion model. Am J Pathol. 2005;166:811–22. pmid:15743793
  78. 78. Richardson K, Weinberg A. Dynamics of regulatory T-cells during pregnancy: effect of HIV infection and correlations with other immune parameters. PloS one. 2011;6:e28172. pmid:22140535
  79. 79. Weinberg A, Song LY, Fenton T, Nachman SA, Read JS, Patterson-Bartlett J, et al. T cell responses of HIV-infected children after administration of inactivated or live attenuated influenza vaccines. AIDS Res Hum Retroviruses. 2010;26:51–9. pmid:20059397
  80. 80. Weinberg A, Muresan P, Fenton T, Richardson K, Dominguez T, Bloom A, et al. High proportions of regulatory B and T cells are associated with decreased cellular responses to pH1N1 influenza vaccine in HIV-infected children and youth (IMPAACT P1088). Human vaccines & immunotherapeutics. 2013;9:957–68.
  81. 81. Hammitt LL, Bartlett JP, Li S, Rahkola J, Lang N, Janoff EN, et al. Kinetics of viral shedding and immune responses in adults following administration of cold-adapted influenza vaccine. Vaccine. 2009;27:7359–66. pmid:19800447
  82. 82. Greenbaum JA, Kotturi MF, Kim Y, Oseroff C, Vaughan K, Salimi N, et al. Pre-existing immunity against swine-origin H1N1 influenza viruses in the general human population. Proceedings of the National Academy of Sciences of the United States of America. 2009;106:20365–70. pmid:19918065
  83. 83. Nayak JL, Fitzgerald TF, Richards KA, Yang H, Treanor JJ, Sant AJ. CD4+ T-cell expansion predicts neutralizing antibody responses to monovalent, inactivated 2009 pandemic influenza A(H1N1) virus subtype H1N1 vaccine. The Journal of infectious diseases. 2013;207:297–305. pmid:23148285
  84. 84. Pedersen GK, Madhun AS, Breakwell L, Hoschler K, Sjursen H, Pathirana RD, et al. T-helper 1 cells elicited by H5N1 vaccination predict seroprotection. The Journal of infectious diseases. 2012;206:158–66. pmid:22551811
  85. 85. Cooper C, Thorne A, Klein M, Conway B, Boivin G, Haase D, et al. Immunogenicity is not improved by increased antigen dose or booster dosing of seasonal influenza vaccine in a randomized trial of HIV infected adults. PloS one. 2011;6:e17758. pmid:21512577
  86. 86. El Sahly HM, Davis C, Kotloff K, Meier J, Winokur PL, Wald A, et al. Higher antigen content improves the immune response to 2009 H1N1 influenza vaccine in HIV-infected adults: a randomized clinical trial. The Journal of infectious diseases. 2012;205:703–12. pmid:22275399
  87. 87. Lagler H, Grabmeier-Pfistershammer K, Touzeau-Romer V, Tobudic S, Ramharter M, Wenisch J, et al. Immunogenicity and tolerability after two doses of non-adjuvanted, whole-virion pandemic influenza A (H1N1) vaccine in HIV-infected individuals. PloS one. 2012;7:e36773. pmid:22629330