| The continued spread of highly pathogenic H5N1 influenza viruses among poultry and wild birds, together with the emergence of drug-resistant variants and the possibility of human-to-human transmission, has spurred attempts to develop an effective vaccine. Inactivated subvirion or whole-virion H5N1 vaccines are safe and have shown promising immunogenicity in clinical trials, but their ability to elicit protective immunity in unprimed human populations remains unknown. Live attenuated vaccines are desirable because they offer the potential for cross-clade immunogenicity, antigen sparing, durable protective antibody responses, especially in young children, and relatively rapid manufacture during a pandemic.In this study, we constructed the H5N1 reassortant viruses by reverse genetics, using published sequences of A/Ann Arbor/6/60 ca (AAca) to generate all genes encoding the internal proteins. The HA and NA genes were from H5N1 virus A/Anhui/2/2005 (AH/05) (clade 2.3.4), or A/Bar-headed goose/3/2005 (BHG/05) (clade 2.2). The multiple basic amino acids at the HA cleavage site, a major virulence motif for H5N1 influenza viruses, were replaced with those found at the HA cleavage site of a nonpathogenic avian influenza virus, as previously described. The resultant viruses (AH/AAca, BHG/AAca) did not cause disease or death in chickens upon intranasal or intravenous administration, and only replicated in the respiratory system and did not cause any disease and death in mice after intranasal inoculation with 106EID50 of the viruses. The cold-adapted (ca) and temperature-sensitive (ts) phenotypes of the AH/AAca virus, attributable to the internal genes of AA/ca, were also confirmed.We first evaluated the immunogenicity and vaccine efficacy of the AH/AAca and BHG/AAca viruses in our mouse model. Groups of 6-week-old female specific-pathogen-free BALB/c mice were inoculated intranasally once or twice (4 weeks apart) with 106EID50 of the AH/AAca or BHG/AAca virus in a 50μL volume. By 4 weeks after the first intranasal immunization, the titers of haemagglutinin inhibition (HI) and neutralization (NT) antibodies against the homologous virus had increased significantly over the pretest values. They also rose sharply after the second vaccination. Antibodies to the heterologous virus were either undetectable (HI) or increased (NT) after the first vaccination, rising significantly after the second immunization.Four weeks after vaccination, we challenged the mice intranasally with a lethal dose (102LD50) of two different H5N1 viruses, AH/05 and BHG/05. In both of the AH/AAca and BHG/AAca vaccinated groups, mice were completely protected from homologous virus challenge in both the single- and two-vaccination groups, virus was not detected in any of the organs tested, and the mice remained healthy over the 2 weeks of observation. By contrast, the virus replicated systemically and was detected in all of the test organs in unvaccinated mice, with death occurring between 6 and 10 days postchallenge. In mice challenged with BHG/05, the virus was detected at low titers (< 2 log10 EID50g-1) in the nasal turbinates of animals that had received a single dose of vaccine AH/AAca, but was undetectable in the organs from mice that were vaccinated twice. All of the vaccinated mice remained healthy during the 2-week observation period, whereas the virus replicated systemically and killed all of the mice within 10 days postchallenge in the unvaccinated group.We next assessed the safety and vaccine efficacy of the AH/AAca reassortant virus in a rhesus macaque (Macaca mulatta) model, we inoculated 2- to 3-year-old female animals (n=8, v1-v8) intranasally with 107EID50 of the AH/AAca virus in a 1-mL volume, twice with a 4-week interval. A control group (n=8, C1-C8) received the same volume of phosphate-buffered saline (PBS). Serum was collected from each animal at 4 weeks after the first vaccination and at 2 weeks after the second vaccination. Intranasal inoculation of the AH/AAca virus was not associated with any serious adverse events.All of the vaccinated macaques had a detectable antibody response by ELISA (enzyme-linked immunosorbent assay) at 4 weeks after the first inoculation. Five of these animals developed HI antibodies to the AH/05 virus, and all had NT antibodies to this virus. Antibody levels in the vaccinated animals increased sharply after the second vaccination. Eight animals had detectable HI antibody to AH/05 at 2 weeks after the second vaccination, while the NT antibody and detectable ELISA antibody also increased sharply, at this interval. Overall, the HI and NT antibody titers against the heterologous virus BHG/05 were 2- to 4-fold lower than those against the AH/05 virus.Three weeks after the second vaccination, animals in each group were challenged with an intratracheal inoculation of 106EID50 of AH/05 virus (n=4) or BHG/05 virus (n=4) in a 3-mL volume. Three days later, two animals from each subgroup were euthanized, and different parts of the respiratory system were collected for virus titration and histologic and immunohistochemical examinations. The remaining animals were observed and euthanized on day 15 postchallenge.Lung tissue from four vaccinated macaques euthanized on day 3 postchallenge with either BHG/05 or AH/05 lacked macroscopic lesions, showed only mild-to-moderate bronchopneumonia with prominent peribronchiolar lymph follicles apparent on microscopic observation, and were free of detectable viral antigen. A spectrum of macroscopic lesions including hyperaemia, exudation, and consolidation were observed in the lung lobes of two unvaccinated control animals challenged with the BHG/05 virus and one challenged with the AH/05 virus. Only prominent swelling of the lymph nodes and tonsil were seen in another control animal challenged with the AH/05 virus. Moderate-to-severe bronchopneumonia with prominent viral antigen expression was a characteristic finding in those nonvaccinated animals.Virus was not isolated from any of the organs tested in the four vaccinated animals challenged with either the AH/05 or BHG/05 virus, but was found at high titers in trachea, bronchus, lung, lymph nodes, and tonsil of the four unvaccinated animals on day 3 postchallenge. Among the eight macaques euthanized on day 15 postchallenge, virus was isolated from tonsil of the two control animals challenged with AH/05 virus, but not from either of the two vaccinated animals. Virus was recovered at a low titer from the nasal swab of a single macaque on day 4 postchallenge and of another on day 6 postchallenge.In summary, our results indicate that two doses of the AH/AAca vaccine are both safe and highly efficacious in nonhuman primates challenged with either homologous or heterologous H5N1 virus. These findings, the first in nonhuman primates, provide a compelling rationale for further testing of the cold-adapted live attenuated H5N1 vaccine in human trials. Beginning in late 2003, outbreaks of H5N1 influenza A virus infection appeared among poultry, and wild birds in numerous countries in Asia and subsequently were reported in Europe and Africa. Despite substantial efforts to control the infection in poultry, H5N1 viruses have continued to evolve and spread, producing human infections in 14 countries, with 236 of the 372 confirmed cases proving fatal. Such findings have sparked great interest in pandemic preparedness as well as in understanding the genetic determinants of influenza virus pathogenicity and the ability of the virus to cross species barriers to mammalian hosts.We recently analyzed a series of H5N1 viruses isolated from healthy ducks in southern China since 1999 and found that these viruses had progressively acquired the ability to replicate and cause disease in mice. In the present study, we explored the genetic basis of this change in virulence by comparing two of the viruses that are genetically similar but differ in their ability to infect mice and have different pathogenicity in mice. A/duck/Guangxi/53/2002 (DKGX/53) is low in mice (MLD50, 6.4), whereas A/duck/Fujian/01/2002 (DKFJ/01) is highly pathogenic (MLD50, 0.9). These two viruses differed by 28 amino acids that were mapped in all gene segments. We used reverse genetics to create a series of single-gene recombinants that contained one gene from DKGX/53 and the remaining seven gene segments from DKFJ/01. We find that the PB2, PB1 or PA genes of DKGX/53 could attenuate DKFJ/01 virus to some extent (10-100 fold), but M gene of DKGX/53 virus attenuated the DKFJ/01 virus over 1000 fold (MLD50, 0.9 vs 4.5). Conversely, the recombinant viruses in the DKGX/53 background, only the one that contains the M gene of DKFJ/01 increased the lethality of the DKGX/53 virus (MLD50, 6.4 vs 3.0). We then generated a series chimeric and mutants and tested in mice, we found that two amino acids at position 30 and 215 in the M1 gene play important roles for the difference of virulence of these two H5N1 avian influenza viruses in mice.The amino acid Asp (D) at the position 30 and Ala (A) at the position 215 in M1 are commonly found in the H5N1 influenza viruses, only the DKGX/53 virus has the amino acid Asn at 30 and Thr at 215 in M1. To investigate if these two amino acids 30N and 215T of M1 gene that found in the DKGX/53 could also attenuate other H5N1 viruses, we introduced them into another two mouse lethal viruses, A/duck/Guangxi/35/01 (DKGX/35) (MLD50, 1.8lgEID50) and A/duck/Guangxi/27/03 (MLD50, 0.6lgEID50), we found the mutant DKGX/35-M1 D30N/A215T and DKGX/27-M1 D30N/A215T only replicated in the lung of mice and was dramatically attenuated in mice (MLD50, 1.8 vs 6.1 and 0.6 vs 4.4).These results first demonstrate that M1 gene plays an important role for the virulence of H5N1 influenza virus, and the amino acid Asn at 30 and Thr at 215 in M1 are important for the attenuation of H5N1 influenza viruses in mouse model, and may be suitable targets for the development of antiviral drugs and attenuated vaccines. |
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