Elicitation of protective immunity against simian immunodeficiency virus infection by a recombinant Sendai virus expressing the Gag protein

The development of effective and safe vaccines against HIV-1 infection is urgently needed to prevent the global dissemination of the virus. To this goal, the establishment of a system to induce efficient and durable antigen expression in vaccinees is one of the key issues [1,2]. We previously established an extremely efficient antigen expression system in mammalian cell cultures using recombinant Sendai virus (SeV) [3–5]. For instance, the amount of HIV-1 gp120 produced in the culture supernatants reached as much as 6.0 mg per litter, the highest attainable level in mammalian cells, including poxvirus vectors [4]. SeV is an enveloped virus with a non-segmented negative sense RNA genome of approximately 15.3 kb and belongs to the family Paramyxoviridae. The virus causes a fatal respiratory disease in mice, but is believed to be non-pathogenic for non-human primates and humans. Here we created a recombinant SeV expressing the Gag antigen of simian immunodeficiency virus (SIV), SeV/SIVgag, and tested its efficacy to protect macaques against SIV challenge infection. Immunization with only the Gag antigen led to a marked reduction in viral load from 105 copies/ml in the control challenged animals to below detectable levels in the immunized animals. SeV/SIVgag was constructed on the background plasmid encoding an attenuated SeV (V[−] SeV) [6] and recovered as described previously [3]. Efficient expression of unprocessed SIV Gag, p55, in SeV/SIVgag-infected CV1 cells was confirmed by Western blotting and immunostaining using a monoclonal anti-SIV Gag p27 antibody. Two cynomolgus macaques (Macaca fascicularis), 1129307001 (Cy01) and 1128206162 (Cy62), were vaccinated intranasally with 108 CIU of SeV/SIVgag three times on weeks 0, 4 and 14. Macaque 1429407002 (Cy02) received a control vaccination with the empty vector virus (SeV/control) on the same schedule. Macaque 1329605029 (Cy29) received no vaccination and was used as a naive control. None of the macaques showed pathological signs, including body weight loss after vaccination. No significant decrease in peripheral CD4 or CD8 cell number was observed, either. The replication of SeV/SIVgag and SeV/control in the respiratory tracts was confirmed by virus isolation into the allantoic fluid of chicken embryos after inoculation with the nasal swabs obtained from Cy02, Cy01 and Cy62 on days 2 and 5 after vaccination. Neither the second nor the third vaccination resulted in detectable SeV in the swabs. By nested reverse transcriptase–polymerase chain reaction using SIV gag-specific primers, SIV gag expression was detected in the nasal swabs in both the SeV/SIVgag vaccinated macaques (Cy01 and Cy62) but not in the SeV/control vaccinated macaque (Cy02). Examination of humoral immune responses by enzyme-linked immunosorbent assay showed a high level induction of plasma anti-SeV antibody in all three macaques. The second and third vaccinations induced rapid increases in their levels, suggesting a boosting effect. On the contrary, no significant induction of plasma anti-SIV Gag p27 antibody was observed. In cytotoxic T lymphocyte assays, SIV Gag-specific cytotoxic T lymphocyte activity was not consistently detected in macaque Cy01 or Cy62, with the exception of one positive result in the latter. The macaques were challenged intravenously with a very large dose (100 TCID50) of SIVmac239 on week 22. All four macaques showed a similar level of plasma SIV load, peaking at week 2 after the challenge (Fig. 1). Thereafter, the plasma SIV RNA level decreased but remained at approximately 105 copies/ml in both the control macaques. In marked contrast, the initial viraemia was followed by significantly lower viral loads (102 to 103 copies/ml) in both the SeV/SIVgag vaccinated macaques. More notably, the plasma viral loads in the immunized macaques eventually fell below the detectable level (100 copies/ml), whereas those in the controls remained as high as approximately 105 copies/ml.Fig. 1.: Plasma simian immunodeficiency virus (SIV) RNA copy numbers (copies/ml) after SIV challenge. Macaques were vaccinated with 108 CIU of SeV vectors three times by intranasal inoculation. They were challenged intravenously with 100 TCID50 of SIVmac239 on week 22 after the first vaccination.