Integrase-Defective Lentiviral Vectors for Delivery of Monoclonal Antibodies against Influenza

Abstract

Delivering rapid protection against infectious agents to non-immune populations is a formidable public health challenge. Although passive immunotherapy is a fast and effective method of protection, large-scale production and administration of monoclonal antibodies (mAbs) is expensive and unpractical. Viral vector-mediated delivery of mAbs offers an attractive alternative to their direct injection. Integrase-defective lentiviral vectors (IDLV) are advantageous for this purpose due to the absence of pre-existing anti-vector immunity and the safety features of non-integration and non-replication. We engineered IDLV to produce the humanized mAb VN04-2 (IDLV-VN04-2), which is broadly neutralizing against H5 influenza A virus (IAV), and tested the vectors' ability to produce antibodies and protect from IAV in vivo. We found that IDLV-transduced cells produced functional VN04-2 mAbs in a time- and dose-dependent fashion. These mAbs specifically bind the hemagglutinin (HA), but not the nucleoprotein (NP) of IAV. VN04-2 mAbs were detected in the serum of mice at different times after intranasal (i.n.) or intramuscular (i.m.) administration of IDLV-VN04-2. Administration of IDLV-VN04-2 by the i.n. route provided rapid protection against lethal IAV challenge, although the protection did not persist at later time points. Our data suggest that administration of mAb-expressing IDLV may represent an effective strategy for rapid protection against infectious diseases.

Keywords: genetic immunization; influenza; integrase-defective lentiviral vector; monoclonal antibody; passive immunity.

Figure 1

Schematic representation of lentiviral plasmids. (A). The lentiviral transfer plasmid pTY2-CMV-huG1-VN04-2 expresses VN04-2 mAbs directed against the H5 hemagglutinin (HA) of influenza A virus (IAV); (B). The packaging plasmid contains all necessary packaging elements and the D116N integrase-inactivating mutation; (C). The plasmid expressing the envelope protein of VSV-G for pseudotyping.

Figure 2

Time- and dose-dependent production of VN04-2 mAbs. Supernatants from 293T cells transduced with increasing doses of IDLV-VN04-2 (corresponding to 50 ng, 125 ng, 500 ng or 1 μg HIV-1 p24) or left untransduced, as a negative control, were collected at (a) 48 h or (b) 72 h post-transduction. Samples were separated on 4–15% SDS–PAGE gels and probed for human IgG by Western blot to determine VN04-2 production. Recombinant VN04-2 (rVN04-2) antibodies were run as a positive control for mAb detection. (c) Aliquots of the same samples depicted in panels (a) and (b) were run on a separate 4–15% SDS–PAGE gel and stained with Coomassie Brilliant Blue to visualize total protein.

Figure 3

VN04-2 mAbs generated from integrase-defective lentiviral vectors (IDLV) bind H5 HA. Supernatants (sups) from cells transduced with IDLV-VN04-2 were used to probe dried nitrocellulose dot plots spotted with recombinant IAV H5 HA protein, or NP as specificity control. Recombinant VN04-2 mAbs (rVN04-2) were used as a positive control for detection of H5 HA. Supernatants from untransduced cells were used as a negative control.

Figure 4

Temporal production of VN04-2 mAbs after IDLV-VN04-2 administration in vivo. (a) Presence of VN04-2 mAbs in the serum of individual mice (n = 3) at 28 days after receiving IDLV-VN04-2 by the intranasal (i.n.) or intramuscular (i.m.) route (200 and 500 RT units, respectively) was measured by Western blot for human IgG. Mixed human sera (H, positive control), serum from an untreated mouse (ctrl, negative control), protein standard marker (M). All in all, 100 µg of total protein was loaded for each sample. Total unstained proteins (bottom) as loading control were visualized using the ChemiDoc MP system (Bio-Rad, Hercules, CA, USA). Groups of 5 mice received (b) i.n. or (c) i.m. administration of IDLV-VN04-2 (250 and 500 RT units, respectively). Levels of serum anti-H5 antibodies before or at 3, 6, 9, 14, 21 and 28 days after IDLV-VN04-2 administration were measured by ELISA. Triangles represent individual mice; lines indicate mean values.

Figure 5

Administration of IDLV-VN04-2 protects from H5 IAV challenge BALB/c mice (n = 5) received IDLV-VN04-2 either by the i.n. or i.m. route (250 or 500 ng RT units, respectively), and IDLV-huG1 by the i.m. route or were left unimmunized as a control (a–c). In a separate experiment, mice received 250 ng RT units of either IDLV-VN04-2 or IDLV-GFP by the i.n. route or were left unimmunized as a control (d–f). Mice were lethally challenged with 2 LD50 of VNH5N1-PR8 IAV 3 days after IDLV administration and monitored for weight loss (a,d) and survival (b,e). Mice that lost ≥20% of their initial body weight were euthanized and counted as dead. Comparison of survival curve (b) p = 0.0096 and (c) p = 0.0004 Log-rank Mantel–Cox test. Protection experiments are representative of three independent replicates. Influenza copy numbers in the lungs were measured by real-time PCR and are shown in (c,f).