Shigeyuki Itamura

Pegylated interferon-|[alpha]| protects type 1 pneumocytes against SARS coronavirus infection in macaques

The primary cause of severe acute respiratory syndrome (SARS) is a newly discovered coronavirus. Replication of this SARS coronavirus (SCV) occurs mainly in the lower respiratory tract, and causes diffuse alveolar damage. Lack of understanding of the pathogenesis of SARS has prevented the rational development of a therapy against this disease. Here we show extensive SCV antigen expression in type 1 pneumocytes of experimentally infected cynomolgus macaques (Macaca fascicularis) at 4 d postinfection (d.p.i.), indicating that this cell type is the primary target for SCV infection early in the disease, and explaining the subsequent pulmonary damage. We also show that prophylactic treatment of SCV-infected macaques with the antiviral agent pegylated interferon-α (IFN-α) significantly reduces viral replication and excretion, viral antigen expression by type 1 pneumocytes and pulmonary damage, compared with untreated macaques. Postexposure treatment with pegylated IFN-α yielded intermediate results. We therefore suggest that pegylated IFN-α protects type 1 pneumocytes from SCV infection, and should be considered a candidate drug for SARS therapy

Cross-Protection against H5N1 Influenza Virus Infection Is Afforded by Intranasal Inoculation with Seasonal Trivalent Inactivated Influenza Vaccine

Abstract Background. Avian H5N1 influenza A virus is an emerging pathogen with the potential to cause substantial human morbidity and mortality. We evaluated the ability of currently licensed seasonal influenza vaccine to confer cross-protection against highly pathogenic H5N1 influenza virus in mice. Methods. BALB/c mice were inoculated 3 times, either intranasally or subcutaneously, with the trivalent inactivated influenza vaccine licensed in Japan for the 2005–2006 season. The vaccine included A/NewCaledonia/20/99 (H1N1), A/NewYork/55/2004 (H3N2), and B/Shanghai/361/2002 viral strains and was administered together with poly(I):poly(C12U) (Ampligen) as an adjuvant. At 14 days after the final inoculation, the inoculated mice were challenged with either the A/HongKong/483/97, the A/Vietnam/1194/04, or the A/Indonesia/6/05 strain of H5N1 influenza virus. Results. Compared with noninoculated mice, those inoculated intranasally manifested cross-reactivity of mucosal IgA and serum IgG with H5N1 virus, as well as both a reduced H5N1 virus titer in nasal-wash samples and increased survival, after challenge with H5N1 virus. Subcutaneous inoculation did not induce a cross-reactive IgA response and did not afford protection against H5N1 viral infection. Conclusions. Intranasal inoculation with annual influenza vaccine plus the Toll-like receptor—3 agonist, poly(I): poly(C12U), may overcome the problem of a limited supply of H5N1 virus vaccine by providing cross-protective mucosal immunity against H5N1 viruses with pandemic potential.

A subcutaneously injected UV-inactivated SARS coronavirus vaccine elicits systemic humoral immunity in mice

Abstract The recent emergence of severe acute respiratory syndrome (SARS) was caused by a novel coronavirus, SARS-CoV. It spread rapidly to many countries and developing a SARS vaccine is now urgently required. In order to study the immunogenicity of UV-inactivated purified SARS-CoV virion as a vaccine candidate, we subcutaneously immunized mice with UV-inactivated SARS-CoV with or without an adjuvant. We chose aluminum hydroxide gel (alum) as an adjuvant, because of its long safety history for human use. We observed that the UV-inactivated SARS-CoV virion elicited a high level of humoral immunity, resulting in the generation of long-term antibody secreting and memory B cells. With the addition of alum to the vaccine formula, serum IgG production was augmented and reached a level similar to that found in hyper-immunized mice, though it was still insufficient to elicit serum IgA antibodies. Notably, the SARS-CoV virion itself was able to induce long-term antibody production even without an adjuvant. Anti-SARS-CoV antibodies elicited in mice recognized both the spike and nucleocapsid proteins of the virus and were able to neutralize the virus. Furthermore, the UV-inactivated virion induced regional lymph node T-cell proliferation and significant levels of cytokine production (IL-2, IL-4, IL-5, IFN-γ and TNF-α) upon restimulation with inactivated SARS-CoV virion in vitro. Thus, a whole killed virion could serve as a candidate antigen for a SARS vaccine to elicit both humoral and cellular immunity.

Characterization of human influenza A (H5N1) virus infection in mice: neuro-, pneumo- and adipotropic infection

Mice (ddY strain, 4 weeks old) were infected intranasally with the H5N1 influenza viruses A/Hong Kong/156/97 (HK156) and A/Hong Kong/483/97 (HK483) isolated from humans. HK156 and HK483 required 200 and 5 p.f.u. of virus, respectively, to give a 50% lethal dose to the mice when the volume of inoculum was set at 10 microl. Both viruses caused encephalitis and severe bronchopneumonia in infected mice. The severity of lung lesions caused by the viruses was essentially similar, whereas HK483 caused more extensive lesions in the brain than did HK156. This was supported by the results of virus titration of organ homogenates, which showed that the virus titres in brains of HK483-infected mice were more than 100-fold higher than those of HK156-infected mice, while those in lungs were almost equivalent. Both viruses were detected in homogenates of the heart, liver, spleen and kidney and blood of the infected mice. Virus antigen was detected by immunohistology in the heart and liver, albeit sporadically, but caused no degenerative change in these organs. The antigen was not detected in the thymus, spleen, pancreas, kidney or gastrointestinal tract. In contrast, virus antigen was found frequently in adipose tissues attached to those organs. The adipose tissues showed severe degenerative change and the virus titres in the tissues were high and comparable to those in lungs. Thus, infection of HK156 and HK483 in our mouse model was pneumo-, neuro- and adipotropic, but not pantropic. Furthermore, HK483 showed higher neurotropism than HK156, which may account for its higher lethality.

WHO recommendations for the viruses used in the 2013–2014 Northern Hemisphere influenza vaccine: Epidemiology, antigenic and genetic characteristics of influenza A(H1N1)pdm09, A(H3N2) and B influenza viruses collected from October 2012 to January 2013 ☆

In February the World Health Organisation (WHO) recommends influenza viruses to be included in influenza vaccines for the forthcoming winter in the Northern Hemisphere. These recommendations are based on data collected by National Influenza Centres (NICs) through the WHO Global Influenza Surveillance and Response System (GISRS) and a more detailed analysis of representative and potential antigenically variant influenza viruses from the WHO Collaborating Centres for Influenza (WHO CCs) and Essential Regulatory Laboratories (ERLs). This article provides a detailed summary of the antigenic and genetic properties of viruses and additional background data used by WHO experts during development of the recommendations of the 2013-2014 Northern Hemisphere influenza vaccine composition.

WHO recommendations for the viruses to be used in the 2012 Southern Hemisphere Influenza Vaccine: Epidemiology, antigenic and genetic characteristics of influenza A(H1N1)pdm09, A(H3N2) and B influenza viruses collected from February to September 2011

In February and September each year the World Health Organisation (WHO) recommends influenza viruses to be included in influenza vaccines for the forthcoming winters in the Northern and Southern Hemispheres respectively. These recommendations are based on data collected by National Influenza Centres (NIC) through the Global Influenza Surveillance and Response System (GISRS) and a more detailed analysis of representative and potential antigenically variant influenza viruses from the WHO Collaborating Centres for Influenza (WHO CCs) and Essential Regulatory Laboratories (ERLs). This article provides a detailed summary of the antigenic and genetic properties of viruses and additional background data used by WHO experts during development of the recommendations for the 2012 Southern Hemisphere influenza vaccine composition.

Productive infection in the murine central nervous system with avian influenza virus A (H5N1) after intranasal inoculation

The H5N1 type of influenza A virus isolated from human patients in 1997 has a characteristic hemagglutinin and was considered to be directly transmitted from birds. Although neuropathogenicity of this virus was not demonstrated in human autopsy cases, some experimental studies using mice have disclosed that this virus infects the central nervous system (CNS) after intranasal inoculation. In this study we focused on the topographical localization of virus-infected cells in the murine CNS after intranasal inoculation. We immunohistochemically examined virus-infected cells in mouse tissues using a rabbit antiserum recognizing the nucleoprotein of influenza A virus. The virus-infected cells appeared initially in the respiratory tract. Thereafter, the virus antigen-positive cells appeared in the olfactory system and the cranial nerve nuclei innervating the facial region. This suggests that this virus is principally transmitted from the nasal cavity to CNS through the cranial nerves. Neurons were frequently infected and glial and ependymal cells were also infected. Transneuronal transmission of the virus might play the important role of viral spread within the CNS.

Homotypic and heterotypic protection against influenza virus infection in mice by recombinant vaccinia virus expressing the haemagglutinin or nucleoprotein gene of influenza virus

Recombinant vaccinia virus expressing the influenza virus haemagglutinin (HA) or nucleoprotein (NP) genes from A/SW/Hong Kong/1/74 (H1N1) under the control of a hybrid promoter containing the P7.5 early promoter element and promoter of the gene encoding the major protein of cowpox virus A type inclusion body was constructed to investigate protective immunity against homologous and heterologous viruses in mice. These recombinant vaccinia viruses produced authentic influenza virus HA and NP in infected cells. The recombinant vaccinia virus-influenza virus HA conferred efficient subtype-specific protection although mice challenged with heterologous influenza viruses underwent initial infection. By contrast, immunization with the recombinant vaccinia-influenza virus NP limited virus multiplication in the lungs against challenge infection with all H1N1 and H3N2 influenza viruses examined, although less efficiently. These results will prompt the re-examination of the possibility of using the recombinant vaccinia virus-influenza virus NP as a cross-protective vaccine.

Human H5N2 Avian Influenza Infection in Japan and the Factors Associated with High H5N2-Neutralizing Antibody Titer

Background H5N2 avian influenza virus infection of humans has not been reported thus far. The first H5N2 avian influenza infection of poultry in Japan occurred in Ibaraki. Methods The subjects were workers at 35 chicken farms in Ibaraki Prefecture, where the H5N2 virus or antibody was isolated from chickens. None of the subjects exhibited influenza symptoms. The H5N2-neutralizing antibody titers of the first and second paired sera samples were compared. To investigate the possible factors for this increase, the H5N2-neutralizing antibody titer (1:40 or more) was calculated for the second samples. A logistic regression analysis was performed to examine the association of these factors with H5N2-neutralizing antibody positivity. Results We performed Wilcoxon matched-pairs signed-ranked test on data collected from 257 subjects, and determined that the H5N2 antibody titers of the second paired sera samples were significantly higher than those of the first samples (P < 0.001). The H5N2 antibody titers of paired sera of 13 subjects without a history of seasonal influenza vaccination within the previous 12 months increased 4-fold or more. The percentage of antibody positivity was 32% for subjects with a history of seasonal influenza vaccination (28% of all subjects) and 13% for those without a history of the same. The adjusted odds ratio of H5N2-neutralizing antibody positivity was 4.6 (95% confidence interval: 1.6-13.7) for those aged over 40 and 3.1 (95% confidence interval: 1.6-6.1) for those with a history of seasonal influenza vaccination within the previous 12 months. Conclusion The results suggest that this may have been the first avian influenza H5N2 infection of poultry to affect humans. A history of seasonal influenza vaccination might be associated with H5N2-neutralizing antibody positivity.

Molecular evolution of hemagglutinin genes of H1N1 swine and human influenza A viruses

SummaryThe hemagglutinin (HA) genes of influenza type A (H1N1) viruses isolated from swine were cloned into plasmid vectors and their nucleotide sequences were determined. A phylogenetic tree for the HA genes of swine and human influenza viruses was constructed by the neighbor-joining method. It showed that the divergence between swine and human HA genes might have occurred around 1905. The estimated rates of synonymous (silent) substitutions for swine and human influenza viruses were almost the same. For both viruses, the rate of synonymous substitution was much higher than that of nonsynonymous (amino acid altering) substitution. It is the case even for only the antigenic sites of the HA. This feature is consistent with the neutral theory of molecular evolution. The rate of nonsynonymous substitution for human influenza viruses was three times the rate for swine influenza viruses. In particular, nonsynonymous substitutions at antigenic sites occurred less frequently in swine than in humans. The difference in the rate of nonsynonymous substitution between swine and human influenza viruses can be explained by the different degrees of functional constraint operating on the amino acid sequence of the HA in both hosts.