CROI 2016 Abstract eBook

Abstract Listing

Poster Abstracts

titre was 6,500 on the IM immunization whereas 100 or lesser in the ID group. A rapid elimination of the expressed antigens at the site of ID immunization underlied the defect in isotype switching as determined by in vivo luciferase analysis and fluorescent microscopy. The antibody response was restored by additional booster immunizations or the co- administration of the TLR-9 inducing CpG ODN 2395 among many TLR agonists examined. Conclusions: Despite the advantages of DC subset targeting and safety, the efficacy of ID immunization may be limited by the rapid removal of the expressed antigen from the skin. Unlike the previous reports, we, for the first time identified the antigen instability underlying the restricted antibody immune response in NF immunization although the T-cell responses were not affected. We additionally suggest ways to restore the compromised B-cell response. Our work has important implication for the optimization of the emerging NF technology.

324 Distinct B-Cell Gene Signatures Define Ability to Respond to H1N1 Vaccine in HIV+ Children Nicola Cotugno 1 ; Lesley De Armas 2 ; Suresh Pallikkuth 2 ; Alberto Cagigi 3 ; Stefano Rinaldi 1 ; Paolo Rossi 1 ; Paolo Palma 1 ; Savita Pahwa 4 1 Bambino Gesù Children’s Hosp, Univ of Rome Tor Vergata, Rome, Italy; 2 Miller Sch of Med, Univ of Miami, Miami, FL, USA; 3 Bambino Gesù Children’s Hosp, Rome, Italy; 4 Univ of Miami Miller Sch of Med, Miami, FL, USA Background: In HIV+ children, the immune response following seasonal or routine vaccinations is often inadequate, thus pointing to the need for predictive biomarkers of response. In this study we hypothesized that gene expression (gExp) of sorted B cell subsets may predict the ability of ART treated, virally suppressed HIV+ children to develop protective immunity to H1N1 following influenza vaccination (TIV). Methods: Blood was collected before (T0) and 21 days after (T1) TIV. HIV+ patients (HIV, n=23) were classified as TIV-responders (R, n=11) based on Ab response to H1N1 (T1/ T0=>4 fold increase), and H1N1-memory B cell ELISpot (>80 spots/10 6 cells at T1) and TIV non-responder (NR, n=12). Healthy Controls (HC, n=10) were all responders. NR and R were similar in terms of viral load, CD4 (# and %), ART regimen and duration. Pre-vaccination samples, sorted by flowcytometry in aliquots of 500 cells each (PBMCs; total B ; and B-cell subsets: Naïve, Na : CD19+CD10-CD27-IgD+; Double Negative, DN : CD19+CD10-CD27-IgD-; Resting Memory, Rem : CD19+CD10-CD27+IgD-CD21+; Activated Memory, AM : CD19+CD10-CD27+IgD-CD21-) were analyzed by multiplexed qPCR for 96 genes (Fluidgm, Biomark). Results: Analysis of variance (ANOVA) among the 4 B cell subsets (NA, DN, Rem, AM) showed higher number of Differentially Expressed Genes (DEGs) (p<0.05) in HIV compared to HC. Collectively 258 DEGs in HIV versus 122 DEGs in HC were found when pairs of B cell subsets were compared. This could be in part due to the largest differences between AM and Rem and the rest of B cell subsets in patients suffering from chronic HIV stimulation. In line with this, a distinct signature of AM was found between HIV and HC (28 DEGs). Although this result was confirmed when R and NR were compared to HC, gExp of AM wasn’t able to discriminate between R and NR (3 DEGs). Conversely, ANOVA of Rem showed 25 DEGs between R and NR. Higher expression of genes involved in adaptive immune response ( APRIL, BTK ), leukocyte activation and BCR signaling ( MTOR , FYN , CD86 ), JAK/STAT signaling ( STAT4, IL6R, IFNAR ) and response to IFN ( IFNAR2, MX1 ) was found in R when compared to NR. Conclusions: Collectively, these results show that Rem, crucial for a potent and specific adaptive response, exhibits a distinct gene signature in HIV who are able to mount a response to TIV not present in NR. Future studies based on sorted subsets are needed to define a multiparameter gExp score able to predict the ability of immune compromised patients to respond to the vaccinations. 325 NextGen Sequencing Defines Response to H1N1 Vaccination in HIV-Infected Individuals Li Yin 1 ; Carina Rodriguez 2 ; JiqiangYao 3 ; Kaifen Chang 1 ; Manju Karki 1 ; FahongYu 1 ; John Sleasman 4 ; Maureen Goodenow 1 1 Univ of Florida, Gainesville, FL, USA; 2 Univ of South Florida, Tampa, FL, USA; 3 Moffitt Cancer Cntr, Tampa, FL, USA; 4 Duke Univ Sch of Med, Durham, NC, USA Background: H1N1 infection is a major co-morbidity among HIV-infected individuals. While H1N1 vaccines offer protection to healthy individuals, responses in HIV-infected individuals are variable. We hypothesized that HIV infection will result in decreased B-cell clonal expansion following H1N1 immunization. Methods: NextGen sequences for immunoglobulin G heavy chain variable region (IgGHV) were generated [median 10,496 (quartile range: 8,600 to 14,405) quality sequences] frommRNA of peripheral blood mononuclear cells and used to quantify IgH biodiversity before and after two doses of H1N1 vaccine administered 4 weeks apart to ten HIV-infected children and adolescents enrolled in IMPAACT P1088. Five individuals demonstrated a ≥4-fold rise from baseline in hemagglutination inhibition titers after the 2 nd dose, while 5 age-matched individuals failed to respond. Sequences from study participants and reference H1N1 neutralizing antibodies (NAb) from GenBank were clustered at 10% genetic distance to identify putative H1N1-specific sequences. Consensus sequences of clusters were queried to IMGT for variable (V) and joining (J) alleles, and length and somatic hypermutation (SHM) in complementary determining regions (CDRs). T test compared two groups with p < 0.05 as significant. Results: Following vaccination, IgGHV biodiversity was reduced by 12% in responders, but not in the nonresponders. Base line sequences from each group clustered with each of 26 unique reference H1N1 NAbs with the same V and J alleles and number of SHM in CDR3 as correspondent H1N1 NAbs. Post vaccination, increases in sequence numbers within multiple clusters was observed in all responders, but in only 2 of 5 nonresponders (p = 0.03). Conclusions: HIV-infected children and adolescents harbor H1N1-specific B-cells that fail to expand in vaccine nonresponders, indicating a failure in oligoclonal expansion among memory B-cells. Reduction in biodiversity and increase in number of sequences clustering with reference H1N1 NAb sequences provide novel and sensitive biomarkers to identify responders to vaccines in general.

Poster Abstracts

124

CROI 2016