Intranasal administration of adjuvant-combined recombinant influenza virus HA vaccine protects mice from the lethal H5N1 virus infection.

Attenuated recombinant H5N1 influenza virus was constructed to develop a safe H5N1 influenza vaccine. The immunogenicity and protective effect of the vaccine prepared from haemagglutinin-modified recombinant H5N1 influenza virus was evaluated in mice intranasally co-administered with cholera toxin B subunit containing a trace amount of holotoxin (CTB*), synthetic double-stranded RNA, poly (I:C) or chitin microparticles (CMP) as adjuvants. Intranasal administration of recombinant H5 HA split vaccine with CTB* or poly(I:C) and/or CMP elicited an immunological response with both anti-H5 HA IgA in the nasal wash and anti-H5 HA IgG antibody in the serum, and showed a protective against lethal H5N1 A/Hong Kong/483/97 (HK483) infection. We also demonstrated that intranasal co-administration of antigen with both poly (I:C) and CMP enhanced the expression of Toll-like receptor (TLR) 3, TLR7 in the spleen. These results indicate that poly (I:C) and CMP are highly effective as mucosal adjuvants for use with the nasal H5N1 vaccine.

Quantitative analysis of Kaposi sarcoma-associated herpesvirus (KSHV) in KSHV-associated diseases.

BACKGROUND: Accurate numbers of copies of Kaposi sarcoma-associated herpesvirus (KSHV) and numbers of virus-infected cells in lesions caused by KSHV-associated diseases are unknown. METHODS: Quantitative polymerase chain reaction (PCR) and computerized imaging of immunohistochemical analysis were performed on pathologic sections of samples from persons with KSHV-associated diseases. RESULTS: Real-time PCR and semiquantitative PCR-Southern blotting demonstrated that DNA extracted from biopsy samples of KS lesions contained approximately 1-2 viral copies/cell. KSHV-associated lymphoma contained 10-50 viral copies/cell. Computerized-image analysis demonstrated that approximately 49% of cells expressed KSHV-encoded latency-associated nuclear antigen in KS biopsy samples. On the basis of results of real-time PCR and computerized-image analysis, the predicted number of viral copies was 3.2 viral copies/cell in KS lesions. Computerized-image analysis also revealed that the expression of open-reading frame (ORF)-50 protein, an immediate early protein of KSHV, was very rare in KS lesions, which implies that they were mainly composed of proliferating cells latently infected with KSHV. In multicentric Castleman disease lesions, 25% of virus-infected cells expressed ORF50 protein, which suggests the frequent lytic replication of KSHV. CONCLUSIONS: Numbers of viral copies and of virus-positive cells vary among KSHV-associated diseases, which suggests different mechanisms of viral pathogenesis. The combination of real-time PCR and computerized-image analysis provides a useful tool for the assessment of the number of viral copies in KSHV-associated diseases.

An ingenious design for peptide vaccines.

For humoral immunization, it may be possible to make effective and safe peptide vaccines for various diseases by selection of proper B-cell epitopes. However, a lack of T-cell epitopes on short peptides, such as those associated with major histocompatibility complex (MHC)-restriction, is a major problem for peptide vaccine development. We propose a solution for the design of peptide vaccines that involves induction of broadly reactive T-cell epitopes via agretopes. The strategy involves positioning multi-agretope type peptides on the N-terminal side of a di-lysine linker and B-cell epitopes on the C-terminal side. The addition of the arginine-glysine-aspartate (RGD)-motif to the N terminus of the peptide enhances its immunogenicity, and enables nasal immunization without adjuvants.

An attenuated LC16m8 smallpox vaccine: analysis of full-genome sequence and induction of immune protection.

The potential threat of smallpox bioterrorism has made urgent the development of lower-virulence vaccinia virus vaccines. An attenuated LC16m8 (m8) vaccine was developed in 1975 from the Lister strain used in the World Health Organization smallpox eradication program but was not used against endemic smallpox. Today, no vaccines can be tested with variola virus for efficacy in humans, and the mechanisms of immune protection against the major intracellular mature virion (IMV) and minor extracellular enveloped virion (EEV) populations of poxviruses are poorly understood. Here, we determined the full-genome sequences of the m8, parental LC16mO (mO), and grandparental Lister (LO) strains and analyzed their evolutionary relationships. Sequence data and PCR analysis indicated that m8 was a progeny of LO and that m8 preserved almost all of the open reading frames of vaccinia virus except for the disrupted EEV envelope gene B5R. In accordance with this genomic background, m8 induced 100% protection against a highly pathogenic vaccinia WR virus in mice by a single vaccination, despite the lack of anti-B5R and anti-EEV antibodies. The immunogenicity and priming efficacy with the m8 vaccine consisting mainly of IMV were as high as those with the intact-EEV parental mO and grandparental LO vaccines. Thus, mice vaccinated with 10(7) PFU of m8 produced low levels of anti-B5R antibodies after WR challenge, probably because of quick clearance of B5R-expressing WR EEV by strong immunity induced by the vaccination. These results suggest that priming with m8 IMV provides efficient protection despite undetectable levels of immunity against EEV.

Secretory IgA antibodies provide cross-protection against infection with different strains of influenza B virus.

This study examined whether secretory IgA (S-IgA) antibodies (Abs) could confer cross-protective immunity against infection with influenza B viruses of antigenically distinct lineages. Wild-type or polymeric Ig receptor (pIgR)-knockout (KO) mice were immunized by infection with different B viruses or by intranasal (i.n.) administration with different inactivated vaccines. Four weeks later mice were challenged with either the B/Ibaraki/2/85 virus, representative of the B/Victoria/2/87 (B/Victoria)-lineage, or B/Yamagata/16/88 virus, representative of the B/Yamagata-lineage. Three days after challenge, nasal wash and serum specimens were assayed for IgA and IgG Abs specific for challenge viral antigens and for protection against challenge viruses. In wild-type mice, B/Ibaraki (or B/Yamagata) cross-reactive IgA Abs were detected at higher levels when infected or immunized with homologous-lineage viruses and at lower levels when infected or immunized with heterologous-lineage viruses. There was a correlation between the amount of nasal cross-reactive IgA Ab and the efficacy of cross-protection with a homologous-lineage virus. In mice lacking the pIgR, nasal cross-protective IgA Abs were only marginally detected in vaccinated mice and an accumulation of IgA in the serum was observed. This reduction of nasal IgA was accompanied by inefficient cross-protection against the B/Ibaraki (or B/Yamagata) virus infection. These results suggest that challenge viral-antigen cross-reactive S-IgA in nasal secretions induced by i.n. infection or vaccination is involved in providing cross-protection against challenge infection with virus within either the B/Victoria- or B/Yamagata-lineage.