H1N1 influenza vaccination in HIV-infected women on effective antiretroviral treatment did not induce measurable antigen-driven proliferation of the HIV-1 proviral reservoir
This study utilized demographic and antibody data and specimens from HIV-1 infected pregnant women who had participated in a Phase II Study to Assess the Safety and Immunogenicity of an Inactivated Swine-Origin H1N1 Influenza Vaccine in HIV-1 Infected Pregnant Women (IMPAACT P1086) [14]. As part of P1086, participants’ blood was collected before and 21 days after the first H1N1 vaccination (high dose, 30 ?g), at which time (21 days post primary vaccination) they received a H1N1 booster. Participants were selected for this substudy based on: (1) ART-suppression of HIV-1 replication (plasma HIV-1 RNA 50 copies/mL, or plasma HIV-1 RNA 500 copies/mL and decreasing in those who started ART approximately at the same time as enrolling into the study), and (2) specimens available for analysis from before and after vaccination. P1086 study participants were not selected based on pre-existing antibody response to H1N1. H1N1 had entered the US beginning ~9 months prior to the P1086 study, therefore P1086 study enrolled a mixture of participants with and without immunity to H1N1 prior to H1N1 vaccination. Utilizing specimens from this H1N1 vaccination trial allowed a unique opportunity to investigate the HIV population immediately before and after a defined antigen stimulus in a mixed population for which H1N1 was a recall antigen for some individuals and a de novo antigen for others. Study endpoints included (1) PBMC DNA load by limited dilution PCR, (2) proportion of monotypic sequences defined as ?2 identical HIV C2-V5 env sequences, and (3) quantification of low-level plasma HIV RNA defined as ?9 c/mL.
DNA was extracted from peripheral blood mononuclear cell (PBMC) samples using a kit (5 Prime ArchivePure DNA Kit; 5 PRIME Inc., Gaithersburg, MD) and quantified using spectrometry (NanoDrop 1000 Spectrophotometer; Thermo Scientific, Waltham, Ma). To quantify HIV-1 DNA, the DNA was serially diluted, triplicates of each dilution underwent PCR to amplify HIV-1 env C2-V5, and the Quality program [15] was used to estimate the number of copies of HIV-1 in the sample, using 1st-round (BH2 [16] and Env6834 (CAG GCC TGT CCA AAA GTA TCC TTT GAG CCA ATT CC), and 2nd-round (DR7 [17] and DR8 [18]) PCR primers, and cycling conditions previously published [19]. To amplify ~20 single viral templates, the DNA from each specimen was diluted to a concentration expected to yield an amplicon from ~30% of PCR; at this concentration 70% of the amplicons are predicted to be from a single viral template. Sequences that appeared in chromatograms to have been derived from more that one template were discarded.
The amplicons were purified (ExoSAP-IT; Affymetrix, Santa Clara, CA) and 2uL of the cleaned product was added to 2uL of primers DR7 and DR8 at concentration of 3.2 pmol for dideoxynucleotide sequencing. Sequences were analyzed using Sequencher program v5.0.1 (Gene Codes Corporation, Ann Arbor, MI) and aligned in Seaview (http://pbil.univlyon1.fr/software/seaview3.html). DIVEIN was used to construct phylogenetic trees and compute divergence of sequences from the most recent common ancestor (MRCA) and sequence diversity [20]. The trees were rooted against reference subtype B sequences except participant 2 who was infected with a complex recombinant and was rooted against a collection of similar sequences in the HIV Sequence Compendium. FigTree (http://tree.bio.ed.ac.uk/software/figtree) was used to graphically represent the phylogenetic trees.
To determine if H1N1 vaccine was associated with low-level viremias, HIV-1 RNA was quantified from subjects’ plasma using previously published methods [21] with the following adaptations. Viral particles from 4.7 mL of plasma (in Beckman OptiSeal Tubes) were pelleted by ultracentrifugation at 175,000 g for 30 min. Afterwards, 3.7 mL of plasma was carefully removed from above the pellet, leaving the 1 mL of plasma overlying the pelleted virions undisturbed. The pellet was resuspended and the HIV RNA was quantified (Abbott RealTime HIV-1 Assay; Abbott Molecular, Abbott Park, Illinois). The calculated lower limit of quantification after concentration of the virions was approximately 9 copies/mL of plasma.
Comparisons of population medians and proportions were performed using Students t-Test for two samples with 2-sided tails (Excel, Microsoft, Redmond, WA).