A bacterially-expressed recombinant envelope protein from Usutu virus induces neutralizing antibodies in rabbits

Background: Recently, an emerging flavivirus, Usutu virus (USUV), has caused an epidemic among birds in Europe, resulting in a massive die-off in Eurasian blackbirds. Currently found only in Europe and Africa, it can be envisioned that Usutu virus will follow the path of other flaviviruses, like West Nile virus and Zika virus, and will spread via its mosquito vectors and bird hosts to other parts of the world. Several cases of human infections by Usutu virus have already been published. Anticipating this spread, development of an efficacious vaccine would be highly desirable. Method: This study describes the production in E. coli, purification, and refolding of a partial USUV envelope protein. Prior to immunization, the protein was characterized using size exclusion chromatography, transmission electron microscopy and dynamic light scattering, showing the limited presence of virus-like structures, indicating that the protein solution is probably a mixture of mono and multimeric envelope proteins. Results: Immunizations of two rabbits with the refolded E-protein fraction, mixed with a strong adjuvant, resulted in the generation of neutralizing antibodies, as evidenced in an in vitro assay. Discussion: The way forward towards a subunit vaccine against Usutu virus infection is discussed.Microscopic imaging and technolog

Characterization of novel polyomaviruses from Bornean and Sumatran orang-utans

Serological screening of sera from orang-utans demonstrated a high percentage of sera that cross-reacted with antigens of the polyomavirus (PyV) simian virus 40. Analysis of archival DNA samples from 71 Bornean and eight Sumatran orang-utans with a broad-spectrum PCR assay resulted in the detection of PyV infections in 11 animals from both species. Sequence analysis of the amplicons revealed considerable differences between the PyVs from Bornean and Sumatran orang-utans. The genome from two PyVs, one from each species, was therefore amplified and sequenced. Both viral genomes revealed a characteristic PyV architecture, but lacked an obvious agnogene. Neighbour-joining analysis positioned the viruses in a large cluster together with viruses from bats, bovines, rodents and several primate PyVs from chimpanzees, African green monkeys, squirrel monkeys and the human Merkel cell PyV

Retrospective serology study of respiratory virus infections in captive great apes

Great apes are extremely sensitive to infections with human respiratory viruses. In this study, we retrospectively analyzed sera from captive chimpanzees, gorillas and orang-utans. More than 1000 sera (403 chimpanzee, 77 gorilla, and 535 orang-utan sera) were analyzed for antibodies to the human respiratory viruses RSV (respiratory syncytial virus, hMPV (human metapneumovirus), H1N1 and H3N2 influenza A viruses, and influenza B virus. In all ape species high seroprevalences were found for RSV, hMPV, and influenza B virus. A high percentage of captive chimpanzees also showed evidence of influenza A H1N1 infections, and had low levels of H3N2 antibodies, while in sera from gorillas and orang-utans antibody levels to influenza A and B viruses were much lower or practically absent. Transmission of respiratory viruses was examined in longitudinal sera of young chimpanzees, and in chimpanzee sera taken during health checks. In young animals isolated cases of influenza infections were monitored, but evidence was found for single introductions followed by a rapid dissemination of RSV and hMPV within the group. Implementation of strict guidelines for handling and housing of nonhuman primates was shown to be an efficient method to reduce the introduction of respiratory infections in colonies of captive animals. RSV seroprevalence rates of chimpanzees remained high, probably due to circulating virus in the chimpanzee colony

Reduction of viral loads by multigenic DNA priming and adenovirus boosting in the SIVmac-macaque model

In this study, we investigated the ability of a multigenic SIV DNA prime/replication-defective adenovirus serotype 5 (rAd/SIV) boost regimen to induce SIV-specific immune responses and protection against intrarectal challenge with SIVmac251 in rhesus macaques. Four rhesus macaques were immunized intramuscularly three times at 8-week intervals with SIV DNA vaccine and boosted once with rAd/STV vaccine Four control macaques received the same amount of mock plasmid DNA and mock adenovirus vector. While the SIV DNA vaccine included plasmids expressing a mutated human IL-12 gene (IL-12N222L) as well as SIVmac239 structural and regulatory genes, the rAd/SIV vaccine contained rAd vectors expressing SIVmac239 genes only. Immunization with SIV DNA vaccine alone induced SIV-specific IFN-gamma ELISPOT responses in only two of four vaccinated macaques, whereas all animals developed SIV-specific T-cell responses and Env- and Tat-specific antibody responses following the rAd/SIV vaccine boost. Upon intrarectal challenge with pathogenic SIVmac251, strong anamnestic Env-specific binding and neutralizing antibody responses were detected in the vaccinated macaques. Overall, the immunized macaques had lower peak and set-point viral loads than control macaques, suggesting that the induced immune responses play a role in the control of viremia. In addition. the loss of CD4(+) T cells was delayed in the vaccinated macaques after challenge. These results indicate that the multigenic DNA prime-adenovirus boost immunization may be a promising approach in developing an effective AIDS vaccine. (c) 2005 Elsevier Ltd. All rights reserved.X1129sciescopu

DNA/long peptide vaccination against conserved regions of SIV induces partial protection against SIVmac251 challenge.

OBJECTIVES: We recently developed a HIVconsv vaccine strategy, consisting of combined conserved regions of HIV-1, to adequately cover viral diversity. To evaluate efficacy in nonhuman primates, an equivalent SIV-derived immunogen SIVconsv was designed and delivered as plasmid DNA or synthetic long peptides. DESIGN: Rhesus macaques lacking protective MHC class I alleles Mamu-A*001 : 01, B*008 : 01, B*017 : 01 were immunized with either SIVconsv synthetic long peptides (S) alone or in combination with plasmid DNA encoding the same conserved regions (D) using SSS or DDSS regimens. METHODS: The SIVconsv synthetic long peptide vaccine consisted of 46 approximately 30-amino acid-long peptides emulsified in Montanide ISA-720 and adjuvanted with pegylated type I interferon and imiquimod. RESULTS: Both SSS and DDSS regimens generated high frequencies of SIV-specific IFN-γ-producing cells comparable with reported adenoviral vector systems. Strong polyfunctional CD4⁺ T-cell and modest CD8⁺ T-cell responses were generated, which were of central memory T-cell phenotype. Furthermore, SIVconsv-specific antibody responses were induced capable of recognizing the Env glycoprotein. Eight weeks after the last immunization, control and SIVconsv-vaccinated animals were challenged intrarectally with 10 MID50 of pathogenic SIVmac251. Two out of six animals in the DDSS group were protected against infection, while all 14 animals in the SSS and two control groups were infected. Vaccine induced SIV-specific IgG responses in mucosal washes prechallenge were highest in the two protected animals. CONCLUSION: This study demonstrates that vaccine-elicited responses towards conserved regions can afford partial protection against a high-dose intrarectal SIVmac251 challenge

Experimental Infection of Rhesus Macaques and Common Marmosets with a European Strain of West Nile Virus

West Nile virus (WNV) is a mosquito-borne flavivirus that infects humans and other mammals. In some cases WNV causes severe neurological disease. During recent years, outbreaks of WNV are increasing in worldwide distribution and novel genetic variants of the virus have been detected. Although a substantial amount of data exists on WNV infections in rodent models, little is known about early events during WNV infection in primates, including humans. To gain a deeper understanding of this process, we performed experimental infections of rhesus macaques and common marmosets with a virulent European WNV strain (WNV-Ita09) and monitored virological, hematological, and biochemical parameters. WNV-Ita09 productively infected both monkey species, with higher replication and wider tissue distribution in common marmosets compared to rhesus macaques. The animals in this study however, did not develop clinical signs of WNV disease, nor showed substantial deviations in clinical laboratory parameters. In both species, the virus induced a rapid CD56dimCD16bright natural killer response, followed by IgM and IgG antibody responses. The results of this study show that healthy rhesus macaques and common marmosets are promising animal models to study WNV-Ita09 infection. Both models may be particularly of use to evaluate potential vaccine candidates or to investigate WNV pathogenesis