Immunogenicity, Safety, And Protective Efficacy Of An Inactivated SARS-associated Coronavirus Vaccine In Rhesus Monkeys

Objective:Establishment of Pathogenicity Model of Severe Acute Respiratory Syndrome Virus Infection on Macaca Rhesus and use the model to evaluate an inactive vaccine candidate against SARS-CoV.Methods:Animals intramuscularly were inoculated with 0. 5μg, 5 μg, 50 μg, 5000 ug of vaccine and PBS as control, then boosted at day 7. On day 22 post-immunization, 12 immunized monkeys and positive control monkeys were challenged with the NS-1 strain of SARS CoV, at a dose of 10~8 plaque-forming units (PFU) per animal. All animals were sacrificed and necropsied on day 15 post-challenge. The humoral and mucosal immune response, in serum cytokine and clinical signs were monitored following the immunity and challenge by ELISA assays for IgG and IgA antibodies, Serum cytokine analysis Neutralization antibody assay and Hematological examination Liver and kidney functions. SARS-CoV was detected in different samples coming from macaca rhesus in different days by virus isolation, immunoflurescence assay and reverse transcription polymerase chain reaction (RT-PCR).Results:1. Serum IgG antibody response: Most of the vaccinated monkeys (11/12, 92%) developed detectable IgG titers (>100) by 7 days post first immunization. All of the animals receiving 50 ug of vaccine exhibited high level antibody titers (≥ 1600) by 21 days post first immunization, the increase of doses appeared to enhance immune responses.2. Mucosal immunology:. Five (41.7%) of the 12 immunized monkeys had SARS-CoV specific secretory IgA (slgA) antibody.3. Cytokine profiles: The concentration of IFN-γ, was significantly higher than those of the pre-immunization levels(P<0.05 and 0.01, respectively), and continued toincrease after second immunization especially in the middle and high groups .The levels of IL-4 produced pre- or post-vaccination in three immunized groups and control group did not exhibit significant changes4. Serum neutralization activity: Higher dosages of vaccine induced higher levels of neutralizing antibody titer.5. X-ray and Pathological findings: Focally thickened and deranged streaks were identified in the lungs of two PBS-control monkeys after challenge, on chest X-ray. No remarkable lesions were shown in the tissues of vaccinated animals and the negative control animals, while there were obvious pathological lesions in the lung and liver of the positive control animals.6. Virological assays: Specific amplification product was obtained by RT-PCR in most of the samples from the 2 PBS-control monkeys challenged by the virus. No virus was recovered from these samples by culture. Immunoflurescence assay were positive in the positive control animals.7. Clinical signs: No systematic complications were observed, while very high dose (5000 ug) caused mild local reactions (indurations, less than 1.6 cm in diameter) post-vaccination. Following live virus challenge, no remarkable changes were observed in all vaccine-immunized animals post challenge. In positive control animal, Monkey #17 developed fever (0.5 °C above the level before challenge) on day 10 post-challenge. The body weight of the monkey # 17 decreased slightly after fever developed, while no obvious change observed in monkeys #18.8. Hematological assays and Serum biochemical indices: After live virus challenge, hematological assays showed reduction in white blood cell (WBC) counts in animals receiving 0.5 \xg of vaccine, but no changes were measured in other immunized groups. In control monkeys, transient drop in WBC count was detected on day 2 post-challenge, but rapid increase with increase of lymphocyte classification were observed on day 5, compared to the average level of all animals pre-immunization. Serum biochemical indices did not show any significant increase up to the day of sacrifice.Conclusion:1. It may be successful to create the model of SARS on rhesus, and show that the animal model can be used to evaluate anti-SARS CoV drugs and vaccines.2. This inactivated vaccine can induce effective concomitant humoral and mucosalimmunity against SARS-CoV infection.3. The results of serum cytokine production in the vaccinated monkeys suggest that the vaccination preferentially induces a Thl-driven response. The findings also imply that the inactivated vaccine candidate may enhance the cellular immune response, including the increased production of IFN-y in monkeys.4. This inactivated vaccine is safe in monkeys and maybe a good candidate for clinical trials.