The SCID/Beige mouse as a model to investigate protection against Yersinia pestis.

In this study, we have shown that severe combined immunodeficient/beige mice reconstituted with hyperimmune Balb/c lymphocytes can be used as a model to demonstrate adoptive and passive protection against plague infection. Reconstitution of severe combined immunodeficient/beige mice was successful in nine out of ten mice as demonstrated by spleen colonisation and sustained circulating immunoglobulin titres. Furthermore, an increase in antibody titre was evident after a booster immunisation of reconstituted mice. Presence of circulating antibody correlated with protection against a systemic plague challenge and indicated that in reconstituted mice adoptive transfer of a functional immune system had occurred. The severe combined immunodeficient/beige mouse was also used to demonstrate passive protection against inhaled and systemic plague infection. The reconstituted severe combined immunodeficient/beige mouse model demonstrating protective immunity against plague will be further developed to identify the immune cell subsets responsible for this protection.

A new improved sub-unit vaccine for plague: the basis of protection.

In this study, we have determined the limit of protection achievable by immunisation with sub-units of Yersinia pestis against the development of plague in an experimental animal model. Co-immunisation with the purified culture-derived F1 and the recombinant V sub-units afforded a greater level of protection than with either sub-unit alone. The protection given by the combined sub-units was several orders of magnitude greater than that afforded by the whole cell killed (Cutter USP) vaccine and was equivalent to that achieved by vaccination with EV76, the live attenuated Y. pestis vaccine strain. However, the combined sub-unit vaccine has clear advantages over the live vaccine in terms of safety of use and absence of side-effects.

Laboratory diagnosis of plague.

In response to an outbreak of a plague-like disease in India, the Public Health Laboratory Service (PHLS) in the UK distributed advice on the isolation and identification of Yersinia pestis. Some of the procedures outlined were evaluated using a number of isolates of Y. pestis, complemented with in-house techniques detecting virulence genes or their products. These laboratory investigations are limited in that they are either only indicative or they take too long (48 hours or more), and thus represent a serious delay to the patient. Successful patient management must be based on a case history, and therapy should be started immediately. Laboratory diagnosis will subsequently rule out most pathogens which cause similar infections, yet will still require confirmation by a reference laboratory.

Characterisation of orbiviruses of the Kemerovo serogroup: comparison of protein and RNA profiles.

Nine viruses of the Kemerovo serogroup (orbivirus genus; family, Reoviridae) isolated from seabird ticks (Ixodes uriae and Ornithodoros maritimus) from eight different geographical locations (four from Scotland, two from Morocco, one from Eire, one from England, one from the Faeroes Islands) were examined. All nine viruses produced unique RNA electropherotypes but showed a 2 : 4 : 3 : 1 grouping of the ten double-stranded RNA segments. The virus-induced proteins were labelled with 35S-methionine in a Vero cell line. Seven of the viruses isolated from Ixodes uriae produced similar profiles of nine major polypeptides. The remaining two viruses, isolated from Ornithodoros maritimus from Morocco, produced profiles in which only five major viral proteins could be readily detected, four of which had similar molecular weights to proteins apparent in the other isolates.

Kinetics of the immune response to the (F1+V) vaccine in models of bubonic and pneumonic plague.

Protection against aerosol challenge with > 300 MLD of Yersinia pestis was observed 7 days after a single immunisation of mice with the F1+V vaccine. At day 60, mice were protected against injected challenge (10(7)MLD) in a vaccine dose-related manner. Recall responses to rV in splenocytes ex vivo at day 98 correlated significantly (p<0.001) with the immunising dose-level of V antigen; no memory response or anti-V serum IgG was detected in killed whole cell vaccine (KWCV) recipients. This may explain the susceptibility of KWCV recipients to aerosol challenge and the enhanced protection conferred by the F1+V sub-unit vaccine, particularly since the anti-F1 responses induced by either vaccine were similarly IgG1-polarised.

Isolation of an English uukuvirus (family Bunyaviridae).

A virus of the Uukuniemi serogroup was isolated from various organs of a moribund kittiwake (Rissa tridactyla) and from ticks (Ixodes uriae) that were feeding on the bird. The kittiwake was found on Marsden beach, north-east England. This is the first virus in the family Bunyaviridae to have been isolated in England and only the second English arthropod-borne virus after louping ill virus, family Togaviridae (Smith & Varma, 1981). The possibility of infection of humans by uukuviruses is discussed.

Investigation of virus-like particles in Leishmania hertigi.

The promastigote forms of an isolate of Leishmania hertigi contain large numbers of virus-like particles (VLPs) in paracrystalline arrays. The VLPs are not present in the amastigote form and it was not possible to purify them from large amounts of the promastigotes.

The proteins expressed in vivo and in vitro by an orbivirus of the Kemerovo serogroup isolated from Ixodes uriae ticks from St. Abb's Head, Scotland.

The genome of FT363 virus, a member of the Kemerovo serogroup of orbiviruses, was shown to consist of 10 segments of dsRNA. Examination of the intracellular proteins expressed by this virus in Vero cells showed the presence of 9 major and several minor products. Similar results were obtained by translation of the virus dsRNA in a rabbit reticulocyte lysate. Comparison of in vivo and in vitro products by immunoprecipitation using polyclonal FT363 ascitic fluid showed a difference in the numbers of polypeptides precipitated. The significance of these results is discussed.

A reovirus from the bedbug, Cimex lectularius.

Large numbers of virus particles were identified by electron microscopy in the epithelial cells of the ventriculus of the bedbug, Cimex lectularius. The morphology of the virus particles and the presence of a segmented double-stranded RNA genome imply that this isolate should be included in the Reoviridae.

The efficacy of ciprofloxacin and doxycycline against experimental tularaemia.

The efficacy of doxycycline and ciprofloxacin against an experimental tularaemia infection was assessed by comparing the median lethal dose (MLD) of Francisella tularensis Schu4 biotype A strain given intraperitoneally to antibiotic-treated and untreated mice. In untreated Porton outbred mice this was <1 cfu. Ciprofloxacin and doxycycline given at 40 mg/kg bd, initiated 48 h before infection and continued for 5 days after infection, afforded protection against intraperitoneal challenges of 3.7 x 10(6) cfu and 6.0 x 10(6) cfu, respectively. Protection was reduced when both antibiotics were given over a similar period at a lower dose regimen (20 mg/kg bd) to 8.8 x 10(5) cfu and 3.5 x 10(2) cfu, respectively. The greater reduction in protection offered by doxycycline was a reflection of the higher in-vitro MIC. Protection also decreased when the antibiotics were initiated 24 h after challenge. The MLD was 3.2 x 10(5) cfu and 1.6 x 10(6) cfu for ciprofloxacin and doxycycline respectively given at 40 mg/kg bd and was reduced further using the lower dose regimen. Overall, 90% of the deaths occurred following the withdrawal of antibiotic, irrespective of the antibiotic dose or type. It was possible to prevent this relapse by extending the antibiotic administration to 10 days after challenge. Ciprofloxacin and doxycycline may be useful for treating tularaemia, although the possibility of relapse should be considered.

Doxycycline or ciprofloxacin prophylaxis and therapy against experimental Yersinia pestis infection in mice.

The efficacy of doxycycline and ciprofloxacin against an experimental plague infection was assessed by comparing the median lethal dose (MLD) of Yersinia pestis in antibiotic-treated and untreated mice. The MLD of Y. pestis GB strain in untreated mice by the intra-peritoneal route was 23 cfu. If ciprofloxacin dosage (20 or 40 mg/kg twice daily) was initiated 48 h before infection, it afforded complete protection against an intra-peritoneal challenge of 5.24 x 10(7) cfu. Ciprofloxacin therapy initiated 24 h post-challenge was less protective, the MLD was raised to 2.0 x 10(5) and 2.2 x 10(5) cfu for 40 and 20 mg/kg respectively. Doxycycline dosage (40 mg/kg twice daily) initiated 48 h prior to infection raised the MLD to 1.6 x 10(4) cfu, but other prophylactic and therapeutic regimes were ineffective against challenges greater than 6.76 x 10(2) cfu. Ciprofloxacin may therefore be a useful antibiotic to consider for the treatment of plague.

In vitro susceptibilities of Burkholderia mallei in comparison to those of other pathogenic Burkholderia spp.

The in vitro antimicrobial susceptibilities of isolates of Burkholderia mallei to 16 antibiotics were assessed and compared with the susceptibilities of Burkholderia pseudomallei and Burkholderia cepacia. The antibiotic susceptibility profile of B. mallei resembled that of B. pseudomallei more closely than that of B. cepacia, which corresponds to their similarities in terms of biochemistry, antigenicity, and pathogenicity. Ceftazidime, imipenem, doxycycline, and ciprofloxacin were active against both B. mallei and B. pseudomallei. Gentamicin was active against B. mallei but not against B. pseudomallei. Antibiotics clinically proven to be effective in the treatment of melioidosis may therefore be effective for treating glanders.

Immunization with live recombinant Salmonella typhimurium aroA producing F1 antigen protects against plague.

An attenuated Salmonella typhimurium strain which expressed the F1 capsular antigen of Yersinia pestis was constructed by transformation of S. typhimurium SL3261 with plasmid pFGAL2a, a derivative of pUC18 which contained the caf1 gene without the leader sequence. The recombinant was used to vaccinate mice intragastrically and intravenously. The immunity induced was able to protect mice against challenge with a virulent strain of plague. Protection correlated with the induction of high titers of immunoglobulin G in serum samples and a specific T-cell response.

Active immunization with recombinant V antigen from Yersinia pestis protects mice against plague.

The gene encoding V antigen from Yersinia pestis was cloned into the plasmid expression vector pGEX-5X-2. When electroporated into Escherichia coli JM109, the recombinant expressed V antigen as a stable fusion protein with glutathione S-transferase. The glutathione S-transferase-V fusion protein was isolated from recombinant E. coli and cleaved with factor Xa to yield purified V antigen as a stable product. Recombinant V antigen was inoculated intraperitoneally into mice and shown to induce a protective immune response against a subcutaneous challenge with 3.74 x 10(6) CFU of virulent Y. pestis. Protection correlated with the induction of a high titer of serum antibodies and a T-cell response specific for recombinant V antigen. These results indicate that V antigen should be a major component of an improved vaccine for plague.

A comparison of Plague vaccine, USP and EV76 vaccine induced protection against Yersinia pestis in a murine model.

The median lethal dose (MLD) of a pathogenic strain of Yersinia pestis was established by three routes of administration in three strains of mouse. There was no significant difference between the MLDs in the different strains of mouse. The MLD by the subcutaneous route in Balb/C and an outbred line was approximately 1 c.f.u.; the MLD following intraperitoneal administration was tenfold higher. There were significant differences in the mean times to death after administration of the challenge by different routes. The relative efficacy of a live attenuated vaccine strain of Y. pestis (EV76) was compared with that of the formaldehyde-killed vaccine (Plague vaccine, USP). EV76 protected against high challenge doses (up to 5.75 x 10(6) MLD), though immunized animals showed side effects of varying severity. The killed vaccine was less effective in terms of dose-protection (deaths occurred after challenge with 4000 MLD) and several of the vaccinated animals suffered sub-lethal, plague-related sequelae to the challenge.

A sub-unit vaccine elicits IgG in serum, spleen cell cultures and bronchial washings and protects immunized animals against pneumonic plague.

In this study, the protection afforded against aerosolized Yersinia pestis by injection of an alhydrogel-adsorbed sub-unit vaccine has been compared with that given by an existing killed whole cell vaccine licensed for human use. The sub-unit vaccine protected mice against exposure to > 10(4) colony-forming units (c.f.u.) of virulent plague organisms (100 LD50 doses), whereas the whole cell vaccine provided only 50% protection against 1.8 x 10(3) c.f.u. In sub-unit vaccinees, IgG to each of the F1 and V antigens contained in the vaccine, was detected in serum, on direct secretion by spleen cells and in broncho-alveolar washings (BAL). In killed whole cell vaccinees, physiologically significant levels of IgG to F1 only were detectable in equivalent samples. Levels of F1-specific IgG in serum, secreted from spleen cells and in BAL were significantly higher (P < 0.01) in sub-unit compared with killed whole cell vaccinees. IgA was not detected in BAL from intra-muscularly dosed sub-unit vaccinees and thus the protection achieved against inhalational challenge with Yersinia pestis is attributed to the induction of systemic immunity to both the F1 and V antigens in the sub-unit vaccine. The enhanced protective efficacy of this sub-unit vaccine over an existing vaccine has been demonstrated in an animal model of pneumonic plague.

Comparison of efficacy of ciprofloxacin and doxycycline against experimental melioidosis and glanders.

Melioidosis and glanders are caused by the closely related species Burkholderia pseudomallei and Burkholderia mallei, respectively. Whereas melioidosis is a significant cause of morbidity in south-east Asia, glanders is extremely rare. The efficacies of ciprofloxacin and doxycycline were assessed against a strain of B. pseudomallei and a strain of B. mallei which were susceptible to both antimicrobials in vitro. Porton outbred mice and Syrian hamsters were given 40 mg/kg of either doxycycline or ciprofloxacin twice daily by sc injection according to one of three regimens: dosing starting 48 h before challenge and continuing for 5 days postchallenge; 5 days' therapy starting immediately after challenge; 5 days' therapy starting 24 h after challenge. Mice were challenged ip with B. pseudomallei 4845 and hamsters were challenged ip with B. mallei 23344. Antimicrobial efficacy was determined by the shift in the median lethal dose (MLD). Ciprofloxacin prophylaxis and immediate therapy both raised the MLD of B. pseudomallei to 4 x 10(6) cfu from 19 cfu in untreated animals, but therapeutic ciprofloxacin only raised the MLD to 180 cfu. The results for doxycycline were similar. Ciprofloxacin prophylaxis raised the MLD of B. mallei 23344 to 4.6 x 10(5) cfu compared with 4 cfu in untreated controls. Immediate therapy raised the MLD to 7.0 x 10(4) cfu and therapy raised the MLD to 1.6 x 10(3) cfu. All regimens of doxycycline protected hamsters against challenges of up to 2 x 10(7) cfu. Despite using a susceptible strain of B. pseudomallei, neither antimicrobial was effective when used therapeutically. The timely administration of either antimicrobial, however, was effective in preventing symptomatic infection. Doxycycline was the superior of the two antimicrobials against experimental glanders although relapse did occur in treated animals approximately 4-5 weeks after challenge.

A single dose sub-unit vaccine protects against pneumonic plague.

In this study, the protection afforded against aerosolised Yersinia pestis by injection of a single dose of an alhydrogel-adsorbed sub-unit vaccine has been compared with that given by an existing killed whole cell vaccine licensed for human use. The sub-unit vaccine, prepared by admixing F1 antigen derived from a Y. pestis cell culture supernatant with recombinant V antigen derived from an E. coli cell lysate, fully protected an outbred strain of mouse against exposure to 10(6) CFU of virulent plague organisms (10(4) mouse lethal doses, MLD). In contrast, the whole cell vaccine provided only 16% protection against the same level of challenge. Furthermore, sub-unit vaccinees were able to clear the bacteria from their lungs post-challenge whereas bacteria were cultured from the lungs of a surviving KWC vaccinee post-challenge. In killed whole cell vaccinees, physiologically significant levels of IgG to F1 only were detectable and the levels of F1-specific IgG in serum and in broncho-alveolar washings were significantly lower (p<0.05) compared with sub-unit vaccinees. In sub-unit vaccinees, an IgG titre to the F1 and V antigens was detected in serum where it was significantly higher (p<0.05) compared with broncho-alveolar washings suggesting that, at the time of challenge, protection is attributable mainly to the combined circulating IgG titre to the F1 and V sub-units. The enhanced protective efficacy of this sub-unit vaccine administered as a single dose compared with an existing vaccine has been demonstrated in an outbred animal model of pneumonic plague.

An IgG1 titre to the F1 and V antigens correlates with protection against plague in the mouse model.

The objective of this study was to identify an immunological correlate of protection for a two-component subunit vaccine for plague, using a mouse model. The components of the vaccine are the F1 and V antigens of the plague-causing organism, Yersinia pestis, which are coadsorbed to alhydrogel and administered intramuscularly. The optimum molar ratio of the subunits was determined by keeping the dose-level of either subunit constant whilst varying the other and observing the effect on specific antibody titre. A two-fold molar excess of F1 to V, achieved by immunizing with 10 micrograms of each antigen, resulted in optimum antibody titres. The dose of vaccine required to protect against an upper and lower subcutaneous challenge with Y. pestis was determined by administering doses in the range 10 micrograms F1 + 10 micrograms V to 0.01 microgram F1 + 0.01 microgram V in a two-dose regimen. For animals immunized at the 1-microgram dose level or higher with F1 + V, an increase in specific IgG1 titre was observed over the 8 months post-boost and they were fully protected against a subcutaneous challenge with 10(5) colony-forming units (CFU) virulent Y. pestis at this time point. However, immunization with 5 micrograms or more of each subunit was required to achieve protection against challenge with 10(7) CFU Y. pestis. A new finding of this study is that the combined titre of the IgG1 subclass, developed to F1 plus V, correlated significantly (P < 0.05) with protection. The titres of IgG1 in vaccinated mice which correlated with 90%, 50% and 10% protection have been determined and provide a useful model to predict vaccine efficacy in man.

Bunyaviridae. Serological relationships.

The family Bunyaviridae comprises 5 genera of lipid-enveloped viruses with trisegmented RNA genomes. One of the genera, Bunyavirus, comprises over a quarter of the known arboviruses. The members of the Hantavirus genus are apparently the exception in that they are not arthropod-borne. The genera are sorted largely on serological grounds. The Nairoviruses, in addition to the presence of the physical marker of a higher molecular weight nucleoprotein, include Congo-Crimean haemorrhagic fever, an apparently isotypic virus of wide distribution.