IDLV-HIV-1 Env vaccination in non-human primates induces affinity maturation of antigen-specific memory B cells

Abstract

HIV continues to be a major global health issue. In spite of successful prevention interventions and treatment methods, the development of an HIV vaccine remains a major priority for the field and would be the optimal strategy to prevent new infections. We showed previously that a single immunization with a SIV-based integrase-defective lentiviral vector (IDLV) expressing the 1086.C HIV-1-envelope induced durable, high-magnitude immune responses in non-human primates (NHPs). In this study, we have further characterized the humoral responses by assessing antibody affinity maturation and antigen-specific memory B-cell persistence in two vaccinated macaques. These animals were also boosted with IDLV expressing the heterologous 1176.C HIV-1-Env to determine if neutralization breadth could be increased, followed by evaluation of the injection sites to assess IDLV persistence. IDLV-Env immunization was associated with persistence of the vector DNA for up to 6 months post immunization and affinity maturation of antigen-specific memory B cells.

Fig. 1

NHP immunization schedule and binding antibody responses. a Six Indian rhesus macaques were primed intramuscularly with 3 × 10⁸ transducing units (TUs) of IDLV-1086.C and boosted twice, at 1-year intervals, first with the same vector IDLV-1086.C and then with IDLV expressing a different envelope 1176.C in an attempt to garner neutralization breadth. Anti-vector immunity in the repeated IDLV immunizations was minimized using two different VSV serotypes for the first and second immunizations. b Anti C.1086 gp140 and c anti 1176.C gp140 protein binding Abs induced by IDLV-Env immunization. The magnitude and durability of anti-Env IgG were measured in 6 animals in plasma. No binding response was detected in plasma samples collected before immunization (week 0). Lines indicate mean values at each time point. Each sample was analyzed in duplicate and the data shown are representative of at least three experiments

Fig. 2

Cross-reactivity of memory B cell-derived antibodies from longitudinal samples against 1086.C and 1176.C gp140 Envs. a Flow plots of 1086.C and 1176.C gp140 staining of Env-reactive memory B cells in the indicated animals at 0, 57, and 109 weeks post immunization. Memory B cells which bound both BV421 (x-axis) and AF647 (y-axis) labeled Envs were defined as double positive (DP) Env-reactive memory B cells. Sorting of DP Env-reactive B cells was performed to ensure sort accuracy. No pre-existing cross-reacting memory B cells were present before vaccination (week 0). b Env reactivity of each individual sorted memory B cell was confirmed in ELISA after in vitro expansion and differentiation into antibody-secreting cells as described in the Methods. The Venn diagrams show the percentage of Env-specific supernatants from Ig-secreting cultured memory B cells that bound to 1086.C gp140 (yellow) and/or 1176.C gp140 (blue) Env. The number of Env-specific memory B-cell supernatants screened for each animal at each time point is indicated below each diagram

Fig. 3

IDLV vaccination induces antibody avidity maturation over time. Purified IgG was isolated from serum and avidity for 1086.C gp120 was tested in SPR. a Binding in response units (RUs) to 1086.C Env gp120 protein over time. b Antibody dissociation rate (k_d in s−1) over time. Data shown are average of two experiments. Lines indicate mean values at each time point

Fig. 4

Linear epitope mapping. The heat map shows gp120 binding over time to multiple HIV-1 strains. Binding intensity is shown for each peptide, corrected with its own background value

Fig. 5

Persistence of antigen-specific circulating memory B cells following IDLV vaccination. a To assess the persistence of the 1086.C-specific circulating memory B cell pool, longitudinal flow cytometry analysis was performed on PBMCs from two vaccinated animals. b Flow plots of gp140 and gp120 (1086, CH505 or RSC3) staining of Env-reactive memory B cells in animal 4430 at 57 weeks post immunization. Memory B cells which bound both BV421 (x-axis) and AF647 (y-axis) labeled Envs were defined as double positive (DP) Env-reactive memory B cells. Sorting of DP Env-reactive B cells was performed to ensure sort accuracy. Memory B cells which bound to the wild-type form of gp120 but not to the d371 mutated Env were defined as differential binders (putative CD4bs Abs)

Fig. 6

IDLV DNA persists at the site of injection at 6 months post immunization. a Total-IDLV DNA PCR and β-actin PCR were performed using the indicated DNA amounts extracted from the muscle biopsies. Genomic DNA (5 ng) extracted from the positive control (CMMT-LV-Neo) corresponding to 5 × 10² cells was also amplified. b Five PCR replicates were performed for each sample to increase the probability of detecting IDLV DNA in all the animals

Fig. 7

Detection of residual integration in NHP 4459 at 1 month post IDLV-1176 immunization. a Alu-PCR for vector integration and total-IDLV PCR were performed on serial dilutions of total DNA extracted from 1-month muscle biopsy of NHP 4459 (left) and genomic DNA extracted from CMMT-LV-Neo positive control (right). b Positive bands in Alu-PCR were extracted and cloned into pCR2.1 TOPO vector and subsequently sequenced using the M13F and M13R primers. The sequence alignment shows that the integrated sequence corresponds to IDLV-1176