Protection against henipavirus infection by use of recombinant adeno-associated virus-vector vaccines

Abstract

Nipah virus (NiV) and Hendra virus (HeV) are closely related, recently emerged paramyxoviruses that are capable of causing considerable morbidity and mortality in several mammalian species, including humans. Henipavirus-specific vaccines are still commercially unavailable, and development of novel antiviral strategies to prevent lethal infections due to henipaviruses is highly desirable. Here we describe the development of adeno-associated virus (AAV) vaccines expressing the NiV G protein. Characterization of these vaccines in mice demonstrated that a single intramuscular AAV injection was sufficient to induce a potent and long-lasting antibody response. Translational studies in hamsters further demonstrated that all vaccinated animals were protected against lethal challenge with NiV. In addition, this vaccine induced a cross-protective immune response that was able to protect 50% of the animals against a challenge by HeV. This study presents a new efficient vaccination strategy against henipaviruses and opens novel perspectives on the use of AAV vectors as vaccines against emergent diseases.

Figure 1.

Follow-up of anti-Nipah virus (NiV) G humoral responses after intramuscular (IM) or intradermal (ID) injection of adeno-associated virus (AAV)–NiV.G vaccine. A and B, Follow-up of the anti-NiV G protein immunoglobulin G (IgG) response by enzyme-linked immunosorbent assay. Results are expressed as the mean optical density at 450 nm (OD_{450 nm}) ± standard error of the mean (SEM; n = 5). The AAV mouse-secreted embryonic alkaline phosphatase (mSEAP) vector, which encodes mSEAP, was used as a negative control. C and D, Measurement of IgG isotype titers 16 weeks after the priming injection. The anti-NiV. G data are presented as mean ± SEM (n = 5). E and F, Measurement of IgG and neutralizing antibody (NAb) titers after a boost injection with AAVrh32.33-NiV.G. The neutralization assay was performed using NiV virus-like particles. IgG titers (E) and NAb titers (F) were measured at 16 weeks after priming injection (Before-Boost) and 4 weeks after the booster (After-Boost). IgG titers are expressed as the reciprocal of the highest dilution that yields an OD_{450 nm}>0.2 ± SEM (n = 5). NAb titers are expressed as the reciprocal of the highest dilution that yields 50% of neutralization ± SEM (n = 5). *P < .05.

Figure 2.

Analysis of adeno-associated virus (AAV) vector persistence and expression in mice. Muscles were harvested from animals (n = 5) injected intramuscularly with AAV8-NiV.G or AAV8-GFP and used for DNA and RNA extraction. A, AAV vector persistence was measured by qRT-PCR, using primers specific to AAV-NiV.G or AAV-GFP DNA. B, AAV-encoded transgene messenger RNA was measured by qRT-PCR, using primers specific to the Nipah virus (NiV) G or the green fluorescent protein complementary DNAs (cDNAs). Samples were normalized by quantifying the number of CD8 (DNA) or GAPDH (cDNA) copies. Results are expressed as the mean number ( ± standard error of the mean) of NiV G DNA and RNA copies per microgram of DNA and RNA, respectively (n = 5). *P < .05.

Figure 3.

AAV-NiV.G vaccination in golden hamsters. A, Follow-up of anti-Nipah virus (NiV) G immunoglobulin G (IgG) response by enzyme-linked immunosorbent assay. Results are expressed as the mean optical density at 450 nm (OD_{450 nm}) ± standard error of the mean (SEM; n = 5). B, Follow-up of anti-NiV neutralizing antibody (NAb) titers. The neutralization assay was performed using NiV virus-like particles. Results are expressed as the reciprocal of the highest dilution that yields 50% of neutralization ± SEM (n = 5). C and D, Survival rates after challenge with henipaviruses in hamsters. Survival outcomes were observed for 29 days after challenge with NiV (C) or Hendra virus (D). Results are expressed as the percentage of animals (n = 6 per group) that survived over time.

Figure 4.

Detection of henipavirus RNA in tissues from immunized and control animals. Brain, lung, and spleen were collected from animals at the time of euthanasia (Tables 1 and 2). RNA was extracted and analyzed by quantitative reverse transcription polymerase chain reaction, using primers hybridizing either to Nipah virus (NiV; A) or Hendra virus (HeV; B) N gene. Samples were normalized by quantifying the number of CD8 (DNA) or GAPDH (complementary DNA) copies. Results are expressed as the relative number of NiV and HeV RNA copies per microgram of RNA. Numbers below the x-axis correspond to individual animals.