Comparison of the levels of human herpesvirus 6 (HHV‐6) DNA and cytokines in the cerebrospinal fluid and serum of children with HHV‐6 encephalopathy

Primary human herpesvirus‐6 (HHV‐6) infection is a common cause of acute sporadic encephalopathy in Japanese children. Occasionally, HHV‐6 is not detected in the cerebrospinal fluid (CSF) of patients with encephalopathy, for example, in those with focal viral encephalitis, such as herpes simplex viral encephalitis. This indicates that HHV‐6 encephalopathy is caused by an indirect mechanism, although this is not fully understood. HHV‐6 DNA, cytokines (interleukin (IL)‐1β, IL‐6, IL‐8, IL‐10, IL‐12 p70, tumor necrosis factor‐α, interferon‐γ), and matrix metalloproteinase‐9 were quantitated in both the CSF and serum of 13 patients with HHV‐6 encephalopathy during the acute phase of the disease. HHV‐6 DNA was detected in the CSF of seven patients with HHV‐6 encephalopathy. The viral DNA concentration was significantly higher in serum than in CSF (mean 1.64 × 10⁴ vs. 5.70 × 10¹ copies/ml; P = 0.003). The lack or low level of viral DNA in the CSF samples suggests that direct invasion of the central nervous system by HHV‐6 is not the main cause of encephalopathy. Additionally, the IL‐10 concentration was significantly higher in serum than in CSF (P < 0.001), whereas there was no significant difference in IL‐6 levels between the CSF and serum samples. Interestingly, the IL‐8 concentration was significantly higher in CSF than in serum (P = 0.038). The distribution of these cytokines differed between CSF and serum. The high CSF concentration of IL‐8 could play an important role in the pathogenesis of encephalopathy. J. Med. Virol. 82:1410–1415, 2010. © 2010 Wiley‐Liss, Inc.     

High level expression of equine herpesvirus 1 glycoproteins D and H and their role in protection against virus challenge in the C3H (H-2K) murine model

N and C-terminal truncated forms of equine herpesvirus 1 (EHV 1) glycoproteins gD and gH were expressed in baculovirus resulting in the production of secreted recombinant proteins. A carboxy-terminal histidine tag was included on each of the genes for protein isolation by nickel affinity chromatography. Recombinant gD was recognized by three gD specific monoclonal antibodies, 20C4, 5H6 and F3132. F3132 is a conformationally dependent monoclonal antibody with virus neutralizing activity. Expression of gH was confirmed by reacting the protein with the gH peptide specific antiserum R319. The truncated gD gene was also expressed as a β-galactosidase fusion protein which was purified from E. coli by nickel affinity chromatography. C3H mice were inoculated with purified recombinant gD or gH or insect cells which had been infected with recombinant baculoviruses. Mice were subsequently challenged with EHV 1. Purified recombinant baculovirus gD provided the most protection and produced high levels of virus neutralizing antibodies. The gD fusion protein was less effective at protecting mice and insect cells infected with either of the recombinant baculoviruses or purified recombinant gH were poor at conferring protection. The results emphasize the importance of using purified proteins in vaccine formulations and of including EHV 1 gD as a component of a subunit vaccine.     

Immunogenicity and protective efficacy of a DNA vaccine encoding the fusion protein of mycobacterium heat shock protein 65(Hsp65) with human interleukin‐2 against Mycobacterium tuberculosis in BALB/c mice

Developing a new generation of vaccines is important for preventing tuberculosis (TB). DNA vaccine is one promising candidate. In this study we evaluated the immunogenicity and protective efficacy of the DNA vaccine encoding the fusion protein of Mycobacterium tuberculosis heat shock protein 65 (Hsp65) with human interleukin‐2 (hIL‐2) in BALB/c mice. We showed that the DNA vaccine pcDNA‐Hsp65‐hIL‐2 could induce high levels of antigen‐specific antibody, IFN‐γ, CD4⁺ and CD8⁺ T cell production. When the immunized mice were infected with M. tuberculosis H37Rv, the organ bacterial loads in the DNA immunized group were significantly reduced compared to those of the saline control group, but the ability to reduce bacteria was not better than for BCG. The histopathology in lungs of the DNA vaccine immunized mice was similar to that of BCG immunized mice, which was obviously ameliorated compared to that of the saline control group. Overall, the DNA vaccine could afford protection against M. tuberculosis infection, though the protection efficacy was not as great as that of conventional BCG.