Robert J. Phillpotts

Rapid identification of flaviviruses based on conserved NS5 gene sequences

Two conserved regions in the sequence of the NS5 gene of Flaviviruses were identified. Primers were designed from the consensus sequence of these regions and were used in a reverse transcription/polymerase chain reaction (RT/PCR) to amplify a region of the central european tick-borne encephalitis virus Kumlinge NS5 gene. The authenticity of the amplified fragment was confirmed by nucleotide sequencing. A band of the expected size was also obtained when this RT/PCR was applied to 13 other flaviviral RNAs. This method may be useful for characterisation of the NS5 genes of flaviviruses and as a potential pan-flavivirus diagnostic tool.

Antibody-dependent enhancement of tick-borne encephalitis virus infectivity.

Fourteen mouse monoclonal antibodies raised against tick-borne encephalitis virus (TBEV) and polyclonal antisera raised against six other flaviviruses, Edge Hill (EHV), Japanese encephalitis (JEV), Langat (LGTV), louping ill (LIV), West Nile (WNV) and yellow fever (YFV), were tested for their ability to enhance the replication of TBEV in cells of the mouse macrophage-like line P388 D1, and for their reactivity in ELISA and haemagglutination inhibition (HI) tests. Irrespective of their specificity for either the 51K or 58K polypeptide present in TBEV-infected cells, 13 of the 14 monoclonal antibodies enhanced the replication of TBEV but not of WNV. The remaining monoclonal antibody, which immunoprecipitated the 58K polypeptide of TBEV enhanced WNV but not TBEV, although it reacted strongly with both viruses in ELISA and HI tests. Only polyclonal antisera against viruses within the tick-borne encephalitis virus complex (TBEV, LGTV and LIV) enhanced TBEV replication, although all the polyclonal antisera reacted with TBEV by ELISA; two (against JEV and WNV) also reacted by HI test and all enhanced the replication of WNV. These findings suggest that with TBEV, enhancement may be TBEV complex-specific rather than flavivirus-specific. Data derived from testing both polyclonal and monoclonal antibodies suggest further that not all antibodies that bind to the envelope glycoprotein of TBEV are able to enhance the replication of TBEV, and that enhancement is epitope-specific.

Interferon-α protects mice against lethal infection with St Louis encephalitis virus delivered by the aerosol and subcutaneous routes

In common with other flaviviruses, there is no specific therapy for St Louis encephalitis (SLE) virus infections. A number of cases have occurred where infection may have been acquired by the aerosol route in laboratory accidents. The recombinant human interferon hybrids IFN-alpha A/D (Roche Laboratories) and IFN-alpha B/D (Ciba-Geigy) have activity in murine models. Given for several days around the time of exposure to the virus or shortly after, these compounds reduce the mortality from SLE virus administered to mice subcutaneously by up to 70%. In an aerosol model of SLE disease, the mortality was reduced to 30-50% compared to 100% in controls, depending on the challenge level of virus. These results suggest that interferon-alpha could be used to reduce the mortality from SLE infection after known exposure to the virus.

Efficacy of HSV-1 ISCOM vaccine in the guinea-pig model of HSV-2 infection

The capability of a herpes simplex virus (HSV)-1 ISCOM vaccine to protect against intravaginal HSV-2 challenge infection in guinea-pigs is described. The protective efficacy of the HSV-1 ISCOM vaccine is compared with that of a purified, aqueous HSV-1 antigen preparation administered using a similar immunization schedule. The results show that female guinea-pigs immunized with two doses of HSV-1 ISCOM vaccine, each consisting of 20 micrograms of protein given 2 weeks apart responded with high ELISA and neutralization antibody titres, and are almost completely protected against the clinical effects of intravaginal challenge with 10(5.2) TCID50 of HSV-2. This cross-protection is significantly greater than that observed in guinea-pigs immunized with a single dose of HSV-1 ISCOM vaccine, two doses of aqueous HSV-1 antigen preparation or two doses of a mock ISCOM vaccine. However, none of the vaccine preparations completely prevented HSV-2 replication following challenge. Western blot and radioimmunoprecipitation of sera from immunized guinea-pigs show the HSV-1 ISCOM vaccine preparation to contain the major HSV-1 glycoproteins. These findings are discussed in relation to the value and potential use of HSV-1 ISCOM vaccine in humans.

Failure of oral 4',6-dichloroflavan to protect against rhinovirus infection in man.

Summary4′, 6-Dichloroflavan, a potent inhibitor of rhinovirus replicationin vitro, was tested in a double-blind placebo controlled volunteer trial for its protective effect against experimental rhinovirus infection. Dichloroflavan was given orally (1 mg/kg, 3 times per day) for 3 doses before, and 13 doses after intransasal challenge with rhinovirus type 9, a type known to be highly sensitive in tissue culture. A total of 63 volunteers were included in the analysis for efficacy.Dichloroflavan did not produce any consistent or significant reduction in quantitative clinical or laboratory evidence of infection, and there was no apparent negative correlation of such data with drug concentrations in plasma. It is concluded that administration of dichloroflavan in the oral formulation tested is not of value in the treatment of human rhinovirus infection.

Passive immunization of mice with monoclonal antibodies raised against tick-borne encephalitis virus

SummaryAdult Balb/c mice were passively immunized with monoclonal antibodies (100 µg/mouse) raised against tick-borne encephalitis (TBE) virus then challenged 24 hours later s. c. with 10 LD50 of TBE virus (Nëudorfl isolate). None of the mice showed evidence of premature death although all except one of the monoclonal antibodies tested are capable of enhancing the infectivity of TBE virus in the Fc receptor-bearing mouse macrophage-like cell line P 388 D1. The ability of monoclonal antibodies to neutralize TBE virusin vitro, and to fix complement was examined, and of these properties only a single monoclonal antibody, which was able to neutralize virus, was also able to protect mice against virus challenge.

A recombinant humanized monoclonal antibody completely protects mice against lethal challenge with Venezuelan equine encephalitis virus

A recombinant humanized antibody to Venezuelan equine encephalitis virus (VEEV) was constructed in a monocistronic adenoviral expression vector with a foot-and-mouth-disease virus-derived 2A self-cleavage oligopeptide inserted between the antibody heavy and light chains. After expression in mammalian cells, the heavy and light chains of the humanized antibody (hu1A4A1IgG1-2A) were completely cleaved and properly dimerized. The purified hu1A4A1IgG1-2A retained VEEV binding affinity and neutralizing activity similar to its parental murine antibody. The half-life of hu1A4A1IgG1-2A in mice was approximately 2 days. Passive immunization of hu1A4A1IgG1-2A in mice (50 microg/mouse) 24 h before or after virulent VEEV challenge provided complete protection, indicating that hu1A4A1IgG1-2A has potent prophylactic and therapeutic effects against VEEV infection.

Alpha interferon as an adenovirus-vectored vaccine adjuvant and antiviral in Venezuelan equine encephalitis virus infection

There are no widely available vaccines or antiviral drugs capable of protecting against infection with Venezuelan equine encephalitis virus (VEEV), although an adenovirus vector expressing VEEV structural proteins protects mice from challenge with VEEV and is potentially a vaccine suitable for human use. This work examines whether alpha interferon (IFN-alpha) could act as an adjuvant for the adenovirus-based vaccine. IFN-alpha was either expressed by a plasmid linked to the adenovirus vaccine or encoded by a separate adenovirus vector administered as a mixture with the vaccine. In contrast to previous reports with other vaccines, the presence of IFN-alpha reduced the antibody response to VEEV. When IFN-alpha was encoded by adenovirus, the lack of a VEEV-specific response was accompanied by an increase in the immune response to the adenovirus vector. IFN-alpha also plays a direct role in defence against virus infection, inducing the expression of a large number of antiviral proteins. Adenovirus-delivered IFN-alpha protected mice from VEEV disease when administered 24 h prior to challenge, but not when administered 6 h post-challenge, suggesting that up to 24 h is required for the development of the IFN-mediated antiviral response.

Clones of MRC-C cells may be superior to the parent line for the culture of 229E-like strains of human respiratory coronavirus

Abstract A clone was selected from the MRC-C continuous heteroploid cell line which was significantly better than MRC-C for the culture of human respiratory coronavirus (HCV) 229E, strain LP. Such clones could prove generally useful for the isolation of HCV-229E from clinical specimens, and for the propagation and assay of laboratory adapted strains.

Biochemical characterization of herpes simplex virus type-1-immunostimulating complexes (ISCOMs): a multi-glycoprotein structure

The preparation and characterization of an immunostimulating complex (ISCOM) preparation containing several HSV-1 glycoproteins, including the major glycoproteins B and D is described. The multi-glycoprotein HSV-1 ISCOM preparation was obtained from a gradient-purified aqueous HSV-1 antigen preparation following extraction from infected cells using a zwitterionic detergent. With polyclonal and monoclonal antibodies to HSV-1 glycoproteins in enzyme-linked immunosorbent assay, SDS-polyacrylamide gel electrophoresis and radioimmunoprecipitation techniques, the HSV-1 ISCOM preparation was shown to contain glycoproteins B, C, D, E, H and I, although further, additional proteins were also present. The DNA content of HSV-1 ISCOMs was determined using a 3H-thymidine labelling method. The protein and DNA contents of the HSV-1 ISCOM preparation are discussed with reference to the potentialities of the preparation as a vaccine for use in human beings.