Comparison of two modern vaccines and previous influenza infection against challenge with an equine influenza virus from the Australian 2007 outbreak

During 2007, large outbreaks of equine influenza (EI) caused by Florida sublineage Clade 1 viruses affected horse populations in Japan and Australia. The likely protection that would be provided by two modern vaccines commercially available in the European Union (an ISCOM-based and a canarypox-based vaccine) at the time of the outbreaks was determined. Vaccinated ponies were challenged with a representative outbreak isolate (A/eq/Sydney/2888-8/07) and levels of protection were compared. A group of ponies infected 18 months previously with a phylogenetically-related isolate from 2003 (A/eq/South Africa/4/03) was also challenged with the 2007 outbreak virus. After experimental infection with A/eq/Sydney/2888-8/07, unvaccinated control ponies all showed clinical signs of infection together with virus shedding. Protection achieved by both vaccination or long-term immunity induced by previous exposure to equine influenza virus (EIV) was characterised by minor signs of disease and reduced virus shedding when compared with unvaccinated control ponies. The three different methods of virus titration in embryonated hens’ eggs, EIV NP-ELISA and quantitative RT-PCR were used to monitor EIV shedding and results were compared. Though the majority of previously infected ponies had low antibody levels at the time of challenge, they demonstrated good clinical protection and limited virus shedding. In summary, we demonstrate that vaccination with current EIV vaccines would partially protect against infection with A/eq/Sydney/2888-8/07-like strains and would help to limit the spread of disease in our vaccinated horse population

Studying longitudinal neutralising antibody levels against Equid herpesvirus 1 in experimentally infected horses using a novel pseudotype based assay

Infection with equid herpesvirus 1 (EHV-1), a DNA virus of the Herpesviridae family represents a significant welfare issue in horses and a great impact on the equine industry. During EHV-1 infection, entry of the virus into different cell types is complex due to the presence of twelve glycoproteins (GPs) on the viral envelope. To investigate virus entry mechanisms, specific combinations of GPs were pseudotyped onto lentiviral vectors. Pseudotyped virus (PV) particles bearing gB, gD, gH and gL were able to transduce several target cell lines (HEK293T/17, RK13, CHO-K1, FHK-Tcl3, MDCK I & II), demonstrating that these four EHV-1 glycoproteins are both essential and sufficient for cell entry. The successful generation of an EHV-1 PV permitted development of a PV neutralisation assay (PVNA). The efficacy of the PVNA was tested by measuring the level of neutralising serum antibodies from EHV-1 experimentally infected horses (n = 52) sampled in a longitudinal manner. The same sera were assessed using a conventional EHV-1 virus neutralisation (VN) assay, exhibiting a strong correlation (r = 0.82) between the two assays. Furthermore, PVs routinely require -80 ◦C for long term storage and a dry ice cold-chain during transport, which can impede dissemination and utilisation in other stakeholder laboratories. Consequently, lyophilisation of EHV-1 PVs was conducted to address this issue. PVs were lyophilised and pellets either reconstituted immediately or stored under various temperature conditions for different time periods. The recovery and functionality of these lyophilised PVs was compared with standard frozen aliquots in titration and neutralisation tests. Results indicated that lyophilisation could be used to stably preserve such complex herpesvirus pseudotypes, even after weeks of storage at room temperature, and that reconstituted EHV-1 PVs could be successfully employed in antibody neutralisation tests

Use of Equine Herpesvirus 1 glycoprotein pseudotyped lentiviral particles for the development of serological tests and assessment of lyophilisation for transport and storage

Equine herpesviruses (EHVs) are enveloped DNA viruses predominantly infecting members of the Equidae family. EHVs primarily cause respiratory disease, however EHV-1 can produce cases of a neurological disease, abortion and neonatal death. Thus, these viruses represent a welfare issue for the equine industry and scientific focus for researchers. EHV-1 exhibits a complex array of 12 glycoproteins on its surface envelope, but it is unclear precisely which are important for virus cell entry and the role of each in host immune response. In order to investigate the contribution of these glycoproteins, pseudotype viruses (PVs) could provide a useful study tool. We have successfully generated functional EHV-1 pseudotyped lentiviruses bearing four glycoproteins, gB, gD, gH and gL (sequences derived from an aborted foetus during a large EHV1 outbreak strain in Normandy, France). PVs were employed in a pseudotype virus neutralisation test (PVNT) to measure levels of specific neutralising antibodies serum samples (n=52) taken longitudinally from experimentally infected ponies, compared with uninfected controls. PVs routinely require -80oC for long term storage and a dry ice cold-chain during transport which can impede dissemination and utilisation in other laboratories. Consequently, we further investigated whether freeze-drying (lyophilisation) of EHV-1 PV could address this issue. PVs were lyophilised and pellets either reconstituted immediately or stored under various temperature conditions, sampling at different timepoints. The recovery and functionality of these lyophilised PVs was compared with standard frozen aliquots in titration and neutralisation tests

Selective expansion of viral variants following experimental transmission of a reconstituted feline immunodeficiency virus quasispecies

Following long-term infection with virus derived from the pathogenic GL8 molecular clone of feline immunodeficiency virus (FIV), a range of viral variants emerged with distinct modes of interaction with the viral receptors CD134 and CXCR4, and sensitivities to neutralizing antibodies. In order to assess whether this viral diversity would be maintained following subsequent transmission, a synthetic quasispecies was reconstituted comprising molecular clones bearing envs from six viral variants and its replicative capacity compared in vivo with a clonal preparation of the parent virus. Infection with either clonal (Group 1) or diverse (Group 2) challenge viruses, resulted in a reduction in CD4+ lymphocytes and an increase in CD8+ lymphocytes. Proviral loads were similar in both study groups, peaking by 10 weeks post-infection, a higher plateau (set-point) being achieved and maintained in study Group 1. Marked differences in the ability of individual viral variants to replicate were noted in Group 2; those most similar to GL8 achieved higher viral loads while variants such as the chimaeras bearing the B14 and B28 Envs grew less well. The defective replication of these variants was not due to suppression by the humoral immune response as virus neutralising antibodies were not elicited within the study period. Similarly, although potent cellular immune responses were detected against determinants in Env, no qualitative differences were revealed between animals infected with either the clonal or the diverse inocula. However, in vitro studies indicated that the reduced replicative capacity of variants B14 and B28 in vivo was associated with altered interactions between the viruses and the viral receptor and co-receptor. The data suggest that viral variants with GL8-like characteristics have an early, replicative advantage and should provide the focus for future vaccine development

Clinical relevance of KRAS mutation detection in metastatic colorectal cancer treated by Cetuximab plus chemotherapy

The predictive value of KRAS mutation in metastatic colorectal cancer (MCRC) patients treated with cetuximab plus chemotherapy has recently been suggested. In our study, 59 patients with a chemotherapy-refractory MCRC treated with cetuximab plus chemotherapy were included and clinical response was evaluated according to response evaluation criteria in solid tumours (RECIST). Tumours were screened for KRAS mutations using first direct sequencing, then two sensitive methods based on SNaPshot and PCR-ligase chain reaction (LCR) assays. Clinical response was evaluated according to gene mutations using the Fisher exact test. Times to progression (TTP) were calculated using the Kaplan–Meier method and compared with log-rank test. A KRAS mutation was detected in 22 out of 59 tumours and, in six cases, was missed by sequencing analysis but detected using the SNaPshot and PCR-LCR assays. Remarkably, no KRAS mutation was found in the 12 patients with clinical response. KRAS mutation was associated with disease progression (P=0.0005) and TTP was significantly decreased in mutated KRAS patients (3 vs 5.5 months, P=0.015). Our study confirms that KRAS mutation is highly predictive of a non-response to cetuximab plus chemotherapy in MCRC and highlights the need to use sensitive molecular methods, such as SNaPshot or PCR-LCR assays, to ensure an efficient mutation detection

‘A phase II study of oral uracil/ftorafur (UFT®) plus leucovorin combined with oxaliplatin (TEGAFOX) as first-line treatment in patients with metastatic colorectal cancer'

This phase II trial was performed to evaluate the efficacy and tolerability of a new combination of Uracil/Ftorafur (UFT®)/leucovorin (LV) and oxaliplatin in patients (pts) with metastatic colorectal cancer (MCRC) who had not received prior chemotherapy for metastatic disease. Between February 2002 and October 2002, 64 patients received UFT® 300 mg m−2 day−1 and LV 90 mg day−1 from day 1 to day 14 combined with oxaliplatin 130 mg m−2 on day 1, every 3 weeks. All patients were evaluable for safety analysis and 58 of 64 patients were eligible for efficacy. Responses were reviewed by an independent review committee. Of the 58 per-protocol defined assessable patients, 1 complete response and 20 partial responses were observed yielding a response rate of 34% (95% CI: 22–47). The median response duration was 8.74 months (range 1.6–14). The median time to progression and the median survival were 5.88 months (95% CI: 4.34–8.21) and 18.2 months (95% CI: 10–20.7), respectively. Diarrhoea and peripheral neuropathy were the most frequent and predictable toxicities. These events were reversible, noncumulative and manageable. Grade 3 diarrhoea occurred in only 11% of the patients. No grade 4 gastrointestinal toxicity was reported in the study. The incidence of grade 3/4 (National Cancer Institute Common Toxicity Criteria 2: NCI-CTC 2) peripheral neuropathy was 15%. Haematological toxicity was of mild to moderate intensity with 10% of the patients with Grade 3/4 neutropenia without any episode of complication. The TEGAFOX regimen, a new combination using UFT®/LV and oxaliplatin every 3 weeks is feasible on an outpatient basis. The combination is safe and active and may offer a promising alternative to the intravenous route. Nevertheless this efficacy results should be confirmed by randomized phase III trials

The Influence of Meteorology on the Spread of Influenza: Survival Analysis of an Equine Influenza (A/H3N8) Outbreak

The influences of relative humidity and ambient temperature on the transmission of influenza A viruses have recently been established under controlled laboratory conditions. The interplay of meteorological factors during an actual influenza epidemic is less clear, and research into the contribution of wind to epidemic spread is scarce. By applying geostatistics and survival analysis to data from a large outbreak of equine influenza (A/H3N8), we quantified the association between hazard of infection and air temperature, relative humidity, rainfall, and wind velocity, whilst controlling for premises-level covariates. The pattern of disease spread in space and time was described using extraction mapping and instantaneous hazard curves. Meteorological conditions at each premises location were estimated by kriging daily meteorological data and analysed as time-lagged time-varying predictors using generalised Cox regression. Meteorological covariates time-lagged by three days were strongly associated with hazard of influenza infection, corresponding closely with the incubation period of equine influenza. Hazard of equine influenza infection was higher when relative humidity was <60% and lowest on days when daily maximum air temperature was 20–25°C. Wind speeds >30 km hour−1 from the direction of nearby infected premises were associated with increased hazard of infection. Through combining detailed influenza outbreak and meteorological data, we provide empirical evidence for the underlying environmental mechanisms that influenced the local spread of an outbreak of influenza A. Our analysis supports, and extends, the findings of studies into influenza A transmission conducted under laboratory conditions. The relationships described are of direct importance for managing disease risk during influenza outbreaks in horses, and more generally, advance our understanding of the transmission of influenza A viruses under field conditions

Induction cisplatin–irinotecan followed by concurrent cisplatin–irinotecan and radiotherapy without surgery in oesophageal cancer: multicenter phase II FFCD trial

A recent phase I study showed that weekly cisplatin, irinotecan and concurrent radiotherapy can be administered with moderate toxicity in patients with oesophageal cancer. Patients with no prior treatment and oesophageal cancer stage I to III, performance status <3, caloric intake >1500 kcal day−1 were included. Chemotherapy, with cisplatin 30 mg m−2 and irinotecan 60 mg m−2, was administered at days 1, 8, 22, 29, and concurrently with radiotherapy at days 43, 50, 64 and 71. Radiotherapy was delivered with 50 or 50.4 Gy in 25 fractions/5 weeks. Forty-three patients were included, 10 stage I, 19 stage II and 14 stage III. Mean age was 59.2 years (range 44–79). A total of 30 out of 43 (69.8%) patients underwent all planned treatment. During induction chemotherapy, 14 severe toxicities of grade 3 or 4 in 10 patients (23.3%) were reported with 57.1% due to haematoxicity. During chemoradiotherapy, 31 severe toxicities of grade 3 or 4 with 64.5% due to haematotoxicity were reported in 18 patients. One toxic death occurred (diarrhoea grade 4). The complete clinical response rate was 58.1% (95% CI: 43.4–72.8%). Overall survival rate at 1 and 2 years was 62.8%, (95% CI, 58.3–77.3%) and 27.9% (95% CI, 13.4–41.3%), respectively. In conclusion, cisplatin–irinotecan–radiotherapy is an active and well-tolerated regimen feasible in out-patients