Impact of in-utero exposure to HIV and latent TB on infant humoral responses.

Introduction: Latent tuberculosis infection (LTBI) is a common coinfection in people living with HIV (PWH). How LTBI and HIV exposure in utero influence the development of infant humoral immunity is not well characterized. To address this question, we assessed the relationship between maternal humoral responses in pregnant women with HIV or with HIV/LTBI on humoral responses in infants to BCG vaccination and TB acquisition. Methods: Plasma samples were obtained from mother infant pairs during pregnancy (14-34 wks gestation) and in infants at 12 and 44 wks of age from the IMPAACT P1078 clinical trial. LTBI was established by Interferon gamma release assay (IGRA). Progression to active TB (ATB) disease was observed in 5 women at various times after giving birth. All infants were BCG vaccinated at birth and tested for IGRA at 44 weeks. Mtb (PPD, ESAT6/CFP10, Ag85A, LAM), HIV (GP120), and Influenza (HA) specific IgG, IgM, and IgA were measured in plasma samples using a bead based Luminex assay with Flexmap 3D. Results: In maternal plasma there were no differences in Mtb-specific antibodies or viral antibodies in relation to maternal IGRA status. ATB progressors showed increases in Mtb-specific antibodies at diagnosis compared to study entry. However, when compared to the non-progressors at entry, progressors had higher levels of Ag85A IgG and reduced ESAT6/CFP10 IgG and LAM IgG, IgM, and IgA1. All infants showed a decrease in IgG to viral antigens (HIV GP120 and HA) from 12 to 44 weeks attributed to waning of maternally transferred antibody titers. However, Mtb-specific (PPD, ESAT6/CFP10, Ag85A, and LAM) IgG and IgM increased from 12 to 44 weeks. HIV and HA IgG levels in maternal and 12-week infant plasma were highly correlated, and ESAT6/CFP10 IgG and LAM IgG showed a relationship between maternal and infant Abs. Finally, in the subset of infants that tested IGRA positive at 44 weeks, we observed a trend for lower LAM IgM compared to IGRA- infants at 44 weeks. Discussion: The results from our study raise the possibility that antibodies to LAM are associated with protection from progression to ATB and support further research into the development of humoral immunity against TB through infection or vaccination.

Accelerated CD8+ T cell maturation in infants with perinatal HIV infection.

In perinatal HIV infection, early antiretroviral therapy (ART) initiation is recommended but questions remain regarding infant immune responses to HIV and its impact on immune development. Using single cell transcriptional and phenotypic analysis we evaluated the T cell compartment at pre-ART initiation of infants with perinatally acquired HIV from Maputo, Mozambique (Towards AIDS Remission Approaches cohort). CD8+ T cell maturation subsets exhibited altered distribution in HIV exposed infected (HEI) infants relative to HIV exposed uninfected infants with reduced naive, increased effectors, higher frequencies of activated T cells, and lower frequencies of cells with markers of self-renewal. Additionally, a cluster of CD8+ T cells identified in HEI displayed gene profiles consistent with cytotoxic T lymphocytes and showed evidence for hyper expansion. Longitudinal phenotypic analysis revealed accelerated maturation of CD8+ T cells was maintained in HEI despite viral control. The results point to an HIV-directed immune response that is likely to influence reservoir establishment.

Longitudinal analysis of innate immune system in infants with perinatal HIV infection until 18 months of age.

With the advent of antiretroviral therapy (ART), perinatal HIV infection is declining globally but prevalence in Sub-Saharan Africa is still greater than other nations. The relationship of HIV replication in early infancy and the developing immune system is not well understood. In this study, we investigated cellular components of the innate immune system including Natural Killer (NK) cells, monocytes, and Dendritic Cells (DC) in a cohort of HIV exposed infected (HEI) and age-matched HIV exposed uninfected (HEU) infants from Mozambique. Study entry was at the first visit after delivery at age 1-2 months for HIV diagnosis and initiation of ART. Phenotypic analysis by multi-parameter flow cytometry revealed an expansion of total NK cells and the dysfunctional, CD56-CD16+, NK cell subset; increased activation in monocytes and DC; and higher levels of inflammatory homing receptor CCR5 on circulating DC subsets in the HEI infants. NKG2A, an inhibitory receptor for NK cytolytic function, was reduced in HEI compared to HEU and positively correlated with pre-ART viral load (VL) while expression of CCR2, the inflammatory homing receptor, on NK was negatively correlated with VL. Other subsets exhibited positive correlations with VL including the frequency of intermediate monocytes amongst total monocytes. Longitudinal analysis of VL indicated suboptimal ART adherence in HEI. Regardless of level of viral suppression achieved, the frequencies of specific innate immune subsets in HEI were normalized to HEU by 18m. These data support the notion that in early life, NK cells play a role in virus control and should be explored for functional attributes that are effective against HIV at this time during development. Overall, our study provides high resolution overview of the innate immune system during perinatal HIV infection.

HIV-1 reservoir evolution in infants infected with clade C from Mozambique.

BACKGROUND: The persistence of HIV-1-infected cells during antiretroviral therapy is well documented but may be modulated by early initiation of antiretroviral therapy in infants. METHODS: Here, we longitudinally analyzed the proviral landscape in nine infants with vertical HIV-1 infection from Mozambique over a median period of 24 months, using single-genome, near full-length, next-generation proviral sequencing. RESULTS: We observed a rapid decline in the frequency of intact proviruses, leading to a disproportional under-representation of intact HIV-1 sequences within the total number of HIV-1 DNA sequences after 12-24 months of therapy. In addition, proviral integration site profiling in one infant demonstrated clonal expansion of infected cells harboring intact proviruses and indicated that viral rebound was associated with an integration site profile dominated by intact proviruses integrated into genic and accessible chromatin locations. CONCLUSION: Together, these results permit rare insight into the evolution of the HIV-1 reservoir in infants infected with HIV-1 and suggest that the rapid decline of intact proviruses, relative to defective proviruses, may be attributed to a higher vulnerability of genome-intact proviruses to antiviral immunity. Technologies to analyze combinations of intact proviral sequences and corresponding integration sites permit a high-resolution analysis of HIV-1 reservoir cells after early antiretroviral treatment initiation in infants.

Immunological age prediction in HIV-infected, ART-treated individuals.

Anti-retroviral therapy (ART) improves life expectancy in people living with HIV (PWH), but it remains unclear how chronic HIV infection affects normal aging of the immune system. Plasma cell-free protein expression and immune phenotypes were assessed in blood from ART treated PWH (19-77yrs, n = 106) and age-matched, HIV-negative controls (HC, n = 103). Using univariate spearman correlation, we identified 277 and 491 age-associated parameters out of a total 1,357 in HC and PWH, respectively. PWH exhibited shared and distinct age-associated immune profiles compared to HC highlighting the effect of HIV infection on immunological aging. Our analysis resulted in an 8-parameter, plasma-detectable inflammatory index that correlated with chronological age of all study participants but was higher overall in PWH. Additionally, predictive modeling for age in HC participants and age-associated parameters generated a 25-parameter signature, IMAP-25, with 70% and 53% accuracy in HC and PWH, respectively. Applying the IMAP-25 signature to immunological data from PWH revealed accelerated aging in PWH by 5.6 yrs. Overall, our results demonstrate that immune signatures, easily monitored in human blood samples, can be used as an indicator of one's 'immunological age' during ART-treated HIV infection and can be applied to other disease states that affect the immune system.

The Effect of JAK1/2 Inhibitors on HIV Reservoir Using Primary Lymphoid Cell Model of HIV Latency.

HIV eradication is hindered by the existence of latent HIV reservoirs in CD4+ T cells. Therapeutic strategies targeting latent cells are required to achieve a functional cure, however the study of latently infected cells from HIV infected persons is extremely challenging due to the lack of biomarkers that uniquely characterize them. In this study, the dual reporter virus HIVGKO was used to investigate latency establishment and maintenance in lymphoid-derived CD4+ T cells. Single cell technologies to evaluate protein expression, host gene expression, and HIV transcript expression were integrated to identify and analyze latently infected cells. FDA-approved, JAK1/2 inhibitors were tested in this system as a potential therapeutic strategy to target the latent reservoir. Latent and productively infected tonsillar CD4+ T cells displayed similar activation profiles as measured by expression of CD69, CD25, and HLADR, however latent cells showed higher CXCR5 expression 3 days post-infection. Single cell analysis revealed a small set of genes, including HIST1-related genes and the inflammatory cytokine, IL32, that were upregulated in latent compared to uninfected and productively infected cells suggesting a role for these molecular pathways in persistent HIV infection. In vitro treatment of HIV-infected CD4+ T cells with physiological concentrations of JAK1/2 inhibitors, ruxolitinib and baricitinib, used in clinical settings to target inflammation, reduced latent and productive infection events when added 24 hr after infection and blocked HIV reactivation from latent cells. Our methods using an established model of HIV latency and lymphoid-derived cells shed light on the biology of latency in a crucial anatomical site for HIV persistence and provides key insights about repurposing baricitinib or ruxolitinib to target the HIV reservoir.

Transcriptional and Immunologic Correlates of Response to Pandemic Influenza Vaccine in Aviremic, HIV-Infected Children.

People living with HIV (PWH) often exhibit poor responses to influenza vaccination despite effective combination anti-retroviral (ART) mediated viral suppression. There exists a paucity of data in identifying immune correlates of influenza vaccine response in context of HIV infection that would be useful in improving its efficacy in PWH, especially in younger individuals. Transcriptomic data were obtained by microarray from whole blood isolated from aviremic pediatric and adolescent HIV-infected individuals (4-25 yrs) given two doses of Novartis/H1N1 09 vaccine during the pandemic H1N1 influenza outbreak. Supervised clustering and gene set enrichment identified contrasts between individuals exhibiting high and low antibody responses to vaccination. High responders exhibited hemagglutination inhibition antibody titers >1:40 post-first dose and 4-fold increase over baseline. Baseline molecular profiles indicated increased gene expression in metabolic stress pathways in low responders compared to high responders. Inflammation-related and interferon-inducible gene expression pathways were higher in low responders 3 wks post-vaccination. The broad age range and developmental stage of participants in this study prompted additional analysis by age group (e.g. <13yrs and ≥13yrs). This analysis revealed differential enrichment of gene pathways before and after vaccination in the two age groups. Notably, CXCR5, a homing marker expressed on T follicular helper (Tfh) cells, was enriched in high responders (>13yrs) following vaccination which was accompanied by peripheral Tfh expansion. Our results comprise a valuable resource of immune correlates of vaccine response to pandemic influenza in HIV infected children that may be used to identify favorable targets for improved vaccine design in different age groups.

An improved method for specific-target preamplification PCR analysis of single blastocysts useful for embryo sexing and high-throughput gene expression analysis.

Gene expression analysis in preimplantation embryos has been used for answering fundamental questions related to development, prediction of pregnancy outcome, and other topics. Limited amounts of mRNA in preimplantation embryos hinders progress in studying the preimplantation embryo. Here, a method was developed involving direct synthesis and specific-target preamplification (STA) of cDNA for gene expression analysis in single blastocysts. Effective cell lysis and genomic DNA removal steps were incorporated into the method. In addition, conditions for real-time PCR of cDNA generated from these processes were improved. By using this system, reliable embryo sexing results based on expression of sex-chromosome linked genes was demonstrated. Calibration curve analysis of PCR results using the Fluidigm Biomark microfluidic platform (Fluidigm, South San Francisco, CA) was performed to evaluate 96 STA cDNA from single blastocysts. In total, 93.75% of the genes were validated. Robust amplification was detected even when STA cDNA from a single blastocyst was diluted 1,024-fold. Further analysis showed that within-assay variation increased when cycle threshold values exceeded 18. Overall, STA quantitative real-time PCR analysis was shown to be useful for analysis of gene expression of multiple specific targets in single blastocysts.

A therapeutic HIV-1 vaccine reduces markers of systemic immune activation and latent infection in patients under highly active antiretroviral therapy.

HIV infection is characterized by chronic immune activation and the establishment of a pool of latently infected cells. Antiretroviral therapy (ART) can suppress viral load to undetectable levels in peripheral blood by standard measure, however immune activation/chronic inflammation and latent infection persist and affect quality of life. We have now shown that a novel therapeutic HIV vaccine consisting of replication-defective HIV (HIVAX), given in the context of viral suppression under ART, can reduce both immune activation/chronic inflammation and latent infection. Immune activation, as measured by percent of CD8 + HLA-DR + CD38 + T cells, approached levels of healthy controls at week 16 following vaccination. Reduced immune activation was accompanied by a reduction in pro-inflammatory cytokines and peripheral α4ß7 + plasmacytoid DC (a marker of mucosal immune activation). Levels of both HIV-1 DNA and 2-LTR circles were reduced at week 16 following vaccination, suggesting HIVAX can impact HIV-1 latency and reduce viral replication. Surprisingly, reduced immune activation/chronic inflammation was accompanied by an increase in the percent of memory CD4 + T cells expressing markers PD-1 and TIM-3. In addition, evaluation of HIV-1 Gag-specific CD4 + T cells for expression of 96 T cell related genes pre- and post-therapy revealed increased expression of a number of genes involved in the regulation of immune activation, T cell activation, and antiviral responses. Overall this study provides evidence that vaccination with HIVAX in subjects under long term antiviral suppression can reduce immune activation/chronic inflammation and latent infection (Clinicaltrials.gov, identifier NCT01428596).

Higher PIK3C2B gene expression of H1N1+ specific B-cells is associated with lower H1N1 immunogenicity after trivalent influenza vaccination in HIV infected children.

Perinatally HIV-infected children (PHIV), despite successful antiretroviral therapy, present suboptimal responses to vaccinations compared to healthy-controls (HC). Here we investigated phenotypic and transcriptional signatures of H1N1-specific B-cells (H1N1-Sp) in PHIV, differentially responding to trivalent-influenza-vaccine (TIV), and HC. Patients were categorized in responders (R) and non-responders (NR) according to hemagglutination-inhibition-assay at baseline and 21 days after TIV. No differences in H1N1-Sp frequencies were found between groups. H1N1-Sp transcriptional analysis revealed a distinct signature between PHIV and HC. NR presented higher PIK3C2B and NOD2 expression compared to R, confirmed by downregulation of PIK3C2B in resting-memory of R after H1N1 in-vitro stimulation. In conclusion this study confirms that qualitative rather than quantitative analyses are needed to characterize immune responses in PHIV. These results further suggest that higher PIK3C2B in H1N1-Sp of NR is associated with lower H1N1 immunogenicity and may be targeted by future modulating strategies to improve TIV responses in PHIV.

Impact of Early Antiretroviral Therapy Initiation on HIV-Specific CD4 and CD8 T Cell Function in Perinatally Infected Children.

Early initiation of antiretroviral therapy (ART) in vertically HIV-infected children limits the size of the virus reservoir, but whether the time of treatment initiation (TI) can durably impact host immune responses associated with HIV infection is still unknown. This study was conducted in PBMC of 20 HIV-infected virally suppressed children on ART (mean age 9.4 y), classified as early treated (ET; age at ART initiation ≤0.5 y, n = 14) or late treated (LT; age at ART initiation 1-10 y, n = 6). Frequencies and functions of Ag-specific CD4 (CD40L+) and CD8 (CD69+) T cells were evaluated by intracellular IL-2, IFN-γ, and TNF-α production with IL-21 in CD4 or CD107a, granzyme B and perforin in CD8 T cells following stimulation with HIV gp140 protein (ENV) or GAG peptides by multiparameter flow cytometry. ET showed a higher proportion of cytokine-producing ENV- and GAG-specific CD4 and CD8 T cells compared with LT. In particular, ET were enriched in polyfunctional T cells. RNA sequencing analysis showed upregulation of immune activation pathways in LT compared with ET. Our results suggest that timing of TI in HIV-infected children has a long-term and measurable impact on the quality of the HIV-specific T cell immune responses and transcriptional profiles of PBMC, reinforcing the importance of early TI.

Implications of Immune Checkpoint Expression During Aging in HIV-Infected People on Antiretroviral Therapy.

Immune checkpoint molecules (ICMs) regulate T cell responses. In chronic viral infections and cancer, where antigens can persistently stimulate the immune system, ICMs can serve as a barrier to effective immune responses. The role of ICMs in the setting of systemic low-grade inflammation as in aging and antiretroviral therapy (ART)-suppressed HIV infection is not known. In this study, we made use of stored samples from the FLORAH cohort of HIV-infected ART-suppressed adults (age range 19-77 years.) and age-matched HIV-uninfected controls. We measured the expression levels of ICMs: PD-1, LAG-3, TIGIT, TIM-3, and 2B4 on resting CD4 and CD8 T cells and maturation subsets. To determine how expression of these molecules can affect T cell function, we stimulated peripheral blood mononuclear cell with HIV Gag or p09/H1N1 antigen and performed intracellular cytokine staining by multiparameter flow cytometry. ICMs were expressed at higher levels in CD8 compared with CD4. PD-1 was the only molecule that remained significantly higher in HIV-infected individuals compared with controls. LAG-3 expression increased with age in CD4 and CD8 T cells. 2B4 expression on CD8 T cells was negatively associated with IL-2 production but showed no effect on CD4 T cell function. TIM-3 expression was negatively associated with IL-21 production in CD4 and CD8 T cells and also negatively correlated with flu vaccine responses in HIV-negative individuals. Taken altogether, this study demonstrates the marked variation in ICM expression in T cells among adults and sheds light on the biology of these molecules and their effects on antigen-specific T cell functions. Overall, our results point to TIM-3 as a potential biomarker for immune function in HIV+ individuals on ART.

Dysfunctional peripheral T follicular helper cells dominate in people with impaired influenza vaccine responses: Results from the FLORAH study.

Antigen-primed cluster of differentiation (CD) 4+ T follicular helper (Tfh) cells interact with B cells in the germinal centers (GCs) of lymph nodes to generate vaccine-induced antibody (Ab) responses. In the circulation, peripheral Tfh (pTfh) cells, a subset of memory CD4 T cells, serve as surrogates for GC Tfh because of several functional and phenotypic similarities between them. We investigated features of H1N1 influenza antigen-specific pTfh (Ag.pTfh) in virologically controlled HIV+ volunteers on antiretroviral therapy (ART) and healthy control (HC) participants selected from a seasonal influenza vaccine responsiveness study. Selection of the participants was made based on age, defined as young (18-40 y) and old (>60 y) and on their classification as a vaccine responder (VR) or vaccine nonresponder (VNR). VRs demonstrated expansion of CD40L+ and CD69+ Ag.pTfh, with induction of intracellular interleukin 21 (IL-21) and inducible costimulator (ICOS) post vaccination; these responses were strongest in young HC VRs and were less prominent in HIV+ individuals of all ages. Ag.pTfh in VNRs exhibited dramatically different characteristics from VRs, displaying an altered phenotype and a cytokine profile dominated by cytokines IL-2, tumor necrosis factor alpha (TNF-α), or IL-17 but lacking in IL-21. In coculture experiments, sorted pTfh did not support the B cell IgG production in VNRs and were predominantly an inflammatory T helper 1 (Th1)/T helper 17 (Th17) phenotype with lower ICOS and higher programmed cell death protein 1 (PD1) expression. Induction of IL-21 and ICOS on Ag.pTfh cells are negatively affected by both aging and HIV infection. Our findings demonstrate that dysfunctional Ag.pTfh cells with an altered IL-21/IL-2 axis contribute to inadequate vaccine responses. Approaches for targeting inflammation or expanding functional Tfh may improve vaccine responses in healthy aging and those aging with HIV infection.

Single Cell Profiling Reveals PTEN Overexpression in Influenza-Specific B cells in Aging HIV-infected individuals on Anti-retroviral Therapy.

Memory B cells (MBC) respond to secondary antigen challenge to protect against infection and to boost immunity following vaccinations. Despite effective treatment, chronic HIV infection disturbs MBCs by reducing numbers and altering functionality due to hyper-activation and increased apoptosis leading to suboptimal antibody responses against common infectious agents. We used single cell gene expression analysis to evaluate antigen-specific memory B cells in peripheral blood of virally-suppressed HIV-infected individuals and healthy controls stratified by serum H1N1 antibody response 3 weeks post-administration of the seasonal trivalent inactivated influenza vaccine. We used a fluorescent probe to isolate influenza H1N1-specific B cells and a multiplexed and targeted RT-PCR approach to measure expression levels of 96 genes involved in B cell activation and function. Gene profiling revealed a 4-gene predictive signature containing the phosphoinositide-3 kinase (PI3K) inhibitor, PTEN, for identifying antigen-specific MBC from HIV-infected individuals compared to healthy controls. Gene co-expression analysis showed that in addition to overexpression of PTEN, there was increased co-expression of type I interferon-associated genes with PTEN on single cell level in HIV compared to controls. This study highlights the persistent defects in MBC from HIV-infected individuals and points to the PI3K signaling pathway as a target for potential immune intervention.

Altered immune cell follicular dynamics in HIV infection following influenza vaccination.

HIV infection changes the lymph node (LN) tissue architecture, potentially impairing the immunologic response to antigenic challenge. The tissue-resident immune cell dynamics in virologically suppressed HIV+ patients on combination antiretroviral therapy (cART) are not clear. We obtained LN biopsies before and 10 to 14 days after trivalent seasonal influenza immunization from healthy controls (HCs) and HIV+ volunteers on cART to investigate CD4+ T follicular helper (Tfh) and B cell dynamics by flow cytometry and quantitative imaging analysis. Prior to vaccination, compared with those in HCs, HIV+ LNs exhibited an altered follicular architecture, but harbored higher numbers of Tfh cells and increased IgG+ follicular memory B cells. Moreover, Tfh cell numbers were dependent upon preservation of the follicular dendritic cell (FDC) network and were predictive of the magnitude of the vaccine-induced IgG responses. Interestingly, postvaccination LN samples in HIV+ participants had significantly (P = 0.0179) reduced Tfh cell numbers compared with prevaccination samples, without evidence for peripheral Tfh (pTfh) cell reduction. We conclude that influenza vaccination alters the cellularity of draining LNs of HIV+ persons in conjunction with development of antigen-specific humoral responses. The underlying mechanism of Tfh cell decline warrants further investigation, as it could bear implications for the rational design of HIV vaccines.

Circulating inflammatory monocytes contribute to impaired influenza vaccine responses in HIV-infected participants.

OBJECTIVE: Antibody responses are often impaired in old age and in HIV-positive (HIV+) infection despite virologic control with antiretroviral therapy but innate immunologic determinants are not well understood. DESIGN: Monocytes and natural killer cells were examined for relationships to age, HIV infection and influenza vaccine responses. METHODS: Virologically suppressed HIV+ (n = 139) and HIV-negative (HIV-) (n = 137) participants classified by age as young (18-39 years), middle-aged (40-59 years) and old (≥60 years) were evaluated preinfluenza and postinfluenza vaccination. RESULTS: Prevaccination frequencies of inflammatory monocytes were highest in old HIV+ and HIV-, with old HIV+ exhibiting higher frequency of integrin CD11b on inflammatory monocytes that was correlated with age, expression of C-C chemokine receptor-2 (CCR2) and plasma soluble tumor necrosis factor receptor-1 (sTNFR1), with inverse correlation with postvaccination influenza H1N1 antibody titers. Higher frequencies of CD11b+ inflammatory monocytes (CD11b(hi), >48.4%) compared with low frequencies of CD11b+ inflammatory monocytes (<15.8%) was associated with higher prevaccination frequencies of total and inflammatory monocytes and higher CCR2 MFI, higher plasma sTNFR1 and CXCL-10 with higher lipopolysaccharide stimulated expression of TNFα and IL-6, concomitant with lower postvaccination influenza antibody titers. In HIV+ CD11b(hi) expressers, the depletion of inflammatory monocytes from peripheral blood mononuclear cells resulted in enhanced antigen-specific CD4+ T-cell proliferation. Immature CD56(hi) natural killer cells were lower in young HIV+ compared with young HIV- participants. CONCLUSION: Perturbations of innate immunity and inflammation signified by high CD11b on inflammatory monocytes are exacerbated with aging in HIV+ and negatively impact immune function involved in Ab response to influenza vaccination.

Impact of aging and HIV infection on serologic response to seasonal influenza vaccination.

OBJECTIVE: To determine influence of age and HIV infection on influenza vaccine responses. DESIGN: Evaluate serologic response to seasonal trivalent influenza vaccine (TIV) as the immunologic outcome in HIV-infected (HIV⁺) and age-matched HIV negative (HIV⁻) adults. METHODS: During 2013-2016, 151 virologically controlled HIV⁺ individuals on antiretroviral therapy and 164 HIV⁻ volunteers grouped by age as young (<40 years), middle aged (40-59 years) and old (≥60 years) were administered TIV and investigated for serum antibody response to vaccine antigens. RESULTS: At prevaccination (T0) titers were in seroprotective range in more than 90% of participants. Antibody titers increased in all participants postvaccination but frequency of classified vaccine responders to individual or all three vaccine antigens at 3-4 weeks was higher in HIV⁻ than HIV⁺ adults with the greatest differences manifesting in the young age group. Of the three vaccine strains in TIV, antibody responses at T2 were weakest against H3N2 with those to H1N1 and B antigens dominating. Among the age groups, the titers for H1N1 and B were lowest in old age, with evidence of an age-associated interaction in HIV⁺ persons with antibody to B antigen. CONCLUSION: Greater frequencies of vaccine nonresponders are seen in HIV⁺ young compared with HIV⁻ adults and the observed age-associated interaction for B antigen in HIV⁺ persons are supportive of the concept of premature immune senescence in controlled HIV infection. High-potency influenza vaccination recommended for healthy aging could be considered for HIV⁺ adults of all ages.

Reevaluation of immune activation in the era of cART and an aging HIV-infected population.

Biological aging is associated with immune activation (IA) and declining immunity due to systemic inflammation. It is widely accepted that HIV infection causes persistent IA and premature immune senescence despite effective antiretroviral therapy and virologic suppression; however, the effects of combined HIV infection and aging are not well defined. Here, we assessed the relationship between markers of IA and inflammation during biological aging in HIV-infected and -uninfected populations. Antibody response to seasonal influenza vaccination was implemented as a measure of immune competence and relationships between IA, inflammation, and antibody responses were explored using statistical modeling appropriate for integrating high-dimensional data sets. Our results show that markers of IA, such as coexpression of HLA antigen D related (HLA-DR) and CD38 on CD4+ T cells, exhibit strong associations with HIV infection but not with biological age. Certain variables that showed a strong relationship with aging, such as declining naive and CD38+ CD4 and CD8+ T cells, did so regardless of HIV infection. Interestingly, the variable of biological age was not identified in a predictive model as significantly impacting vaccine responses in either group, while distinct IA and inflammatory variables were closely associated with vaccine response in HIV-infected and -uninfected populations. These findings shed light on the most relevant and persistent immune defects during virological suppression with antiretroviral therapy.

Paradoxical aging in HIV: immune senescence of B Cells is most prominent in young age.

Combination antiretroviral therapies (cART)can lead to normal life expectancy in HIV-infected persons, and people aged >50 yrs represent the fastest growing HIV group. Although HIV and aging are independently associated with impaired humoral immunity, immune status in people aging with HIV is relatively unexplored. In this study influenza vaccination was used to probe age associated perturbations in the B cell compartment of HIV-negative "healthy controls" (HC) and virologically controlled HIV-infected participants on cART (HIV) (n=124), grouped by age as young (<40 yrs), middle-aged (40-59yrs) or old (>60 yrs). H1N1 antibody response at d21 post-vaccination correlated inversely with age in both HC and HIV. Immunophenotyping of cryopreserved PBMC demonstrated increased frequencies of double negative B cells and decreased plasmablasts in old compared to young HC. Remarkably, young HIV were different from young HC but similar to old HC in B cell phenotype, influenza specific spontaneous (d7) or memory (d21) antibody secreting cells. We conclude that B cell immune senescence is a prominent phenomenon in young HIV in comparison to young HC, but distinctions between old HIV and old HC are less evident though both groups manifest age-associated B cell dysfunction.

Induction of IL21 in Peripheral T Follicular Helper Cells Is an Indicator of Influenza Vaccine Response in a Previously Vaccinated HIV-Infected Pediatric Cohort.

HIV-infected patients of all ages frequently underperform in response to seasonal influenza vaccination, despite virologic control of HIV. The molecular mechanisms governing this impairment, as well as predictive biomarkers for responsiveness, remain unknown. This study was performed in samples obtained prevaccination (T0) from HIV-infected children who received the 2012-2013 seasonal influenza vaccine. Response status was determined based on established criterion for hemagglutination inhibition titer; participants with a hemagglutination titer ≥1:40 plus a ≥4-fold increase over T0 at 3 wk postvaccination were designated as responders. All children had a history of prior influenza vaccinations. At T0, the frequencies of CD4 T cell subsets, including peripheral T follicular helper (pTfh) cells, which provide help to B cells for developing into Ab-secreting cells, were similar between responders and nonresponders. However, in response to in vitro stimulation with influenza A/California/7/2009 (H1N1) Ag, differential gene expression related to pTfh cell function was observed by Fluidigm high-density RT-PCR between responders and nonresponders. In responders, H1N1 stimulation at T0 also resulted in CXCR5 induction (mRNA and protein) in CD4 T cells and IL21 gene induction in pTfh cells that were strongly associated with H1N1-specific B cell responses postvaccination. In contrast, CD4 T cells of nonresponders exhibited increased expression of IL2 and STAT5 genes, which are known to antagonize peripheral Tfh cell function. These results suggest that the quality of pTfh cells at the time of immunization is important for influenza vaccine responses and provide a rationale for targeted, ex vivo Ag-driven molecular profiling of purified immune cells to detect predictive biomarkers of the vaccine response.