The availability of a universal influenza vaccine able to induce broad cross-reactive immune responses against diverse influenza viruses would provide an alternative to currently available strain-specific vaccines. We evaluated the ability of vectors based on modified vaccinia virus Ankara (MVA) expressing conserved influenza proteins to protect mice against lethal challenge with multiple influenza subtypes.
Mice were immunized with MVA vectors expressing H5N1-derived nucleoprotein (NP), the stem region of hemagglutinin (HA), matrix proteins 1 and 2 (M1 and M2), the viral polymerase basic protein 1 (PB1), or the HA stem fused to a quadrivalent matrix protein 2 extracellular domain (M2e). Immunized mice were challenged with lethal doses of H5N1, H7N1 or H9N2 virus and monitored for disease symptoms and weight loss. To investigate the influence of previous exposure to influenza virus on protective immune responses induced by conserved influenza proteins, mice were infected with pandemic H1N1 virus (H1N1pdm09) prior to immunization and subsequently challenged with H5N1 virus. Antibody and T cell responses were assessed by ELISA and flow cytometry, respectively.
MVA vectors expressing NP alone, or co-expressed with other conserved influenza proteins, protected mice against lethal challenge with H5N1, H7N1 or H9N2 virus. Pre-exposure to H1N1pdm09 increased protective efficacy against lethal H5N1 challenge. None of the other conserved influenza proteins provided significant levels of protection against lethal challenge. NP-expressing vectors induced high numbers of influenza-specific CD4+ and CD8+ T cells and high titer influenza-specific antibody responses. Higher influenza-specific CD4+ T cell responses and NP-specific CD8+ T cell responses were associated with increased protective efficacy.
MVA vectors expressing influenza NP protect mice against lethal challenge with H5N1, H7N1 and H9N2 viruses by a mechanism involving influenza-specific CD4+ and CD8+ T cell responses.
For clinical development of a novel multivalent OspA vaccine against Lyme borreliosis, serological assays are required which can be used to establish immune correlates of protection against infection with Borrelia.
Four assays (an OspA IgG ELISA, a competitive inhibition (CI) ELISA, a Borrelia surface-binding (SB) assay and a Borrelia killing assay) were used to evaluate the correlation between immune responses induced by rOspA 1/2 (a chimeric immunogen containing protective epitopes from OspA serotypes 1 and 2), and protective immunity against infection by B. burgdorferi s.s. (OspA-1) and B. afzelii (OspA-2). Mice were immunized with OspA 1/2 doses ranging from 0.3 ng to 100 ng, to induce a range of OspA antibody titers, and exposed to needle challenge with B. burgdorferi s.s. or tick challenge with B. afzelii. Receiver operator characteristics (ROC) curves were constructed for each assay, and the area under the curve (AUC), sensitivity, specificity and Youden Index were calculated. Potential cutoff antibody titers which could be used as correlates of vaccine-induced protection were derived from the maximum Youden Index.
Immunization with OspA-1/2 provided dose-dependent protection against infection with B. burgdorferi s.s. and B. afzelii. Antibody responses detected by all four assays were highly significantly correlated with protection from infection by either B. burgdorferi s.s. (p<0.0001 to 0.0062) or B. afzelii (p<0.0001). ROC analyses of the diagnostic effectiveness of each assay showed the AUC to range between 0.95 and 0.79, demonstrating that all assays distinguish well between infected and non-infected animals. Based on sensitivity, specificity and AUC, the OspA IgG ELISA and SB assays best discriminated between infected and non-infected animals.
All four assays differentiate well between Borrelia-infected and non-infected animals. The relatively simple, high throughput IgG ELISA would be suitable to establish immune correlates of protection for the novel OspA vaccine in clinical trials.
Baseline data were collected to inform the adoption, implementation and institutionalisation phases of a completely smoke free campus policy at a large Western Australian university with a diverse student and staff community.
An online survey was randomly emailed to staff and students to measure the attitudes towards and the acceptability and enforcement of the policy prior to implementation. In total, 969 respondents completed the survey.
General attitudes towards smoking were negative. While smokers, ex-smokers and non-smokers were supportive of smoke free policy on campus, 65.7% of respondents felt the campus should be completely smoke free. Respondents indicated a smoke free policy should be stringently enforced. The majority of respondents reported that they had been exposed to second-hand smoke on campus (n = 768; 79.5%).
Theory of Organisational Change provides a useful framework to support the implementation of the completely smoke free policy in the University setting. The implementation process needs to consider the broad range of issues associated with implementing a completely smoke free policy and address issues such as safety of smokers, ensuring smokers are not marginalised and ensuring a comprehensive program is implemented. These baseline findings can be used to advocate for the implementation of a comprehensive range of strategies that recognise the addictive nature of tobacco smoking and address attitude and behaviour change, environmental adaptations and effective implementation of the policy. Administration should consider smokers and non-smokers when policy is implemented.
Smoke-free policy; Smoking attitudes; University settings; Young people; Theory of Organisational Change
The inhabitants of the mammalian gut are not always relatively benign commensal bacteria but may also include larger and more parasitic organisms, such as worms and protozoa. At some level, all these organisms are capable of interacting with each other. We found that successful establishment of the chronically infecting parasitic nematode Trichuris muris in the large intestine of mice is dependent on microflora and coincident with modulation of the host immune response. By reducing the number of bacteria in the host animal, we significantly reduced the number of hatched T. muris eggs. Critical interactions between bacteria (microflora) and parasites (macrofauna) introduced a new dynamic to the intestinal niche, which has fundamental implications for our current concepts of intestinal homeostasis and regulation of immunity.
The introduction of C-C chemokine receptor type-5 (CCR5) antagonists as antiretroviral therapy has led to the need to study HIV co-receptor tropism in different HIV-1 subtypes and geographical locations. This study was undertaken to evaluate HIV-1 co-receptor tropism in the developing world where non-B subtypes predominate, in order to assess the therapeutic and prophylactic potential of CCR5 antagonists in these regions.
HIV-1-infected patients were recruited into this prospective, cross-sectional, epidemiologic study from HIV clinics in South Africa, Uganda and India. Patients were infected with subtypes C (South Africa, India) or A or D (Uganda). HIV-1 subtype and co-receptor tropism were determined and analyzed with disease characteristics, including viral load and CD4+ and CD8+ T cell counts.
CCR5-tropic (R5) HIV-1 was detected in 96% of treatment-naïve (TN) and treatment-experienced (TE) patients in India, 71% of TE South African patients, and 86% (subtype A/A1) and 71% (subtype D) of TN and TE Ugandan patients. Dual/mixed-tropic HIV-1 was found in 4% of Indian, 25% of South African and 13% (subtype A/A1) and 29% (subtype D) of Ugandan patients. Prior antiretroviral treatment was associated with decreased R5 tropism; however, this decrease was less in subtype C from India (TE: 94%, TN: 97%) than in subtypes A (TE: 59%; TN: 91%) and D (TE: 30%; TN: 79%). R5 virus infection in all three subtypes correlated with higher CD4+ count.
R5 HIV-1 was predominant in TN individuals with HIV-1 subtypes C, A, and D and TE individuals with subtypes C and A. Higher CD4+ count correlated with R5 prevalence, while treatment experience was associated with increased non-R5 infection in all subtypes.
co-receptor; tropism; HIV-1 subtype; HIV-1 clade; CCR5 (R5); CXCR4 (X4); cellular factors
A Vero cell culture–derived seasonal influenza vaccine provides consistently high levels of protection against cell culture–confirmed infection over a complete influenza season. Influenza symptoms are also less severe and of shorter duration in individuals who become infected despite vaccination.
Background. Current knowledge of the consistency of protection induced by seasonal influenza vaccines over the duration of a full influenza season is limited, and little is known about the clinical course of disease in individuals who become infected despite vaccination.
Methods. Data from a randomized double-blind placebo-controlled clinical trial undertaken in healthy young adults in the 2008–2009 influenza season were used to investigate the weekly cumulative efficacy of a Vero cell culture–derived influenza vaccine. In addition, the duration and severity of disease in vaccine and placebo recipients with cell culture–confirmed influenza infection were compared.
Results. Vaccine efficacy against matching strains was consistently high (73%–82%) throughout the study, including the entire period of the influenza season during which influenza activity was above the epidemic threshold. Vaccine efficacy was also consistent (68%–83%) when calculated for all strains, irrespective of antigenic match. Vaccination also ameliorated disease symptoms when infection was not prevented. Bivariate analysis of duration and severity showed a significant amelioration of myalgia (P = .003), headache (P = .025), and fatigue (P = .013) in infected vaccinated subjects compared with placebo. Cough (P = .143) and oropharyngeal pain (P = .083) were also reduced in infected vaccinated subjects.
Conclusions. A Vero cell culture–derived influenza vaccine provides consistently high levels of protection against cell culture–confirmed infection by seasonal influenza virus and significantly reduces the duration and severity of disease in those individuals in which infection is not prevented.
Clinical Trials Registration. ClinicalTrials.gov NCT00566345.
Vero cell culture-derived whole-virus H5N1 vaccines have been extensively tested in clinical trials and consistently demonstrated to be safe and immunogenic; however, clinical efficacy is difficult to evaluate in the absence of wide-spread human disease. A lethal mouse model has been utilized which allows investigation of the protective efficacy of active vaccination or passive transfer of vaccine induced sera following lethal H5N1 challenge.
We used passive transfer of immune sera to investigate antibody-mediated protection elicited by a Vero cell-derived, non-adjuvanted inactivated whole-virus H5N1 vaccine. Mice were injected intravenously with H5N1 vaccine-induced rodent or human immune sera and subsequently challenged with a lethal dose of wild-type H5N1 virus.
Passive transfer of H5N1 vaccine-induced mouse, guinea pig and human immune sera provided dose-dependent protection of recipient mice against lethal challenge with wild-type H5N1 virus. Protective dose fifty values for serum H5N1 neutralizing antibody titers were calculated to be ≤1∶11 for all immune sera, independently of source species.
These data underpin the confidence that the Vero cell culture-derived, whole-virus H5N1 vaccine will be effective in a pandemic situation and support the use of neutralizing serum antibody titers as a correlate of protection for H5N1 vaccines.
The recent advent of murine leukaemia virus (MLV)-based replication-competent retroviral (RCR) vector technology has provided exciting new tools for gene delivery, albeit the advances in vector efficiency which have been realized are also accompanied by a set of fresh challenges. The expression of additional transgene sequences, for example, increases the length of the viral genome, which can lead to reductions in replication efficiency and in turn to vector genome instability. This necessitates efforts to analyse the rate and mechanism of recombinant emergence during the replication of such vectors to provide data which should contribute to improvements in RCR vector design.
In this study, we have performed detailed molecular analyses on packaged vector genomes and proviral DNA following propagation of MLV-based RCR vectors both in cell culture and in pre-formed subcutaneous tumours in vivo. The effects of strain of MLV, transgene position and host cell type on the rate of emergence of vector recombinants were quantitatively analysed by applying real-time PCR and real-time RT-PCR assays. Individual mutants were further characterized by PCR, and nucleotide sequence and structural motifs associated with these mutants were determined by sequencing. Our data indicate that virus strain, vector design and host cell influence the rate of emergence of predominating vector mutants, but not the underlying recombination mechanisms in vitro. In contrast, however, differences in the RNA secondary structural motifs associated with sequenced mutants emerging in cell culture and in solid tumours in vivo were observed.
Our data provide further evidence that MLV-based RCR vectors based on the Moloney strain of MLV and containing the transgene cassette in the 3' UTR region are superior to those based on Akv-MLV and/or containing the transgene cassette in the U3 region of the LTR. The observed discrepancies between the data obtained in solid tumours in vivo and our own and previously published data from infected cells in vitro demonstrates the importance of evaluating vectors designed for use in cancer gene therapy in vivo as well as in vitro.
Rous sarcoma virus (RSV) can be used for the simple generation of high-titer replication-competent retroviral (RCR) vectors. Retroviruses undergo frequent genomic recombination, however, and vectors with reduced replication kinetics are rapidly overgrown by mutant forms. Vector design is hence critical to vector efficacy. In this study, two different designs of RSV-based RCR vectors were evaluated. Vectors in which transgene expression was facilitated by the v-src splice acceptor were revealed to have greatly reduced replication kinetics and genomic stability in comparison to vectors in which transgene expression was mediated by an internal ribosome entry site in the 3′ untranslated region.
Combivir is a fixed dose combination tablet of two antiretroviral drugs; zidovudine and lamivudine, used in the treatment of HIV-1 infection. AZT was the first antiretroviral used in clinical trials and the addition of lamivudine improved its effectiveness. With the introduction of highly active antiretroviral therapy in the form of a combination of three drugs including two nucleoside analogues, Combivir became the gold standard nucleoside ‘backbone’ until very recently. Combivir was the first combination agent and simplified HIV therapy greatly. The introduction of newer fixed dose combinations with the advantage of once daily dosing and improved tolerability and toxicity profiles has made Combivir a less popular choice in treatment naïve individuals needing to start therapy.
Combivir; zidovudine; lamivudine; antiretroviral; HAART; HIV
The limited efficiency of in vivo gene transfer by replication-deficient retroviral vectors remains an obstacle to achieving effective gene therapy for solid tumors. One approach to circumvent this problem is the use of replication-competent retroviral vectors. However, the application of such vectors is at a comparatively early stage and the effects which virus strain, transgene cassette position, and target cell can exert on vector spread kinetics, genomic stability, and transgene expression levels remain to be fully elucidated. Thus, in this study a panel of vectors allowing the investigation of different design features on an otherwise genetically identical background were analyzed with respect to these readout parameters in cultures of both murine and human cells and in preformed tumors in nude mice. The obtained data revealed that (i) Moloney murine leukemia virus (Mo-MLV)-based vectors spread with faster kinetics, drive higher levels of transgene expression, and are more stable than equivalent Akv-MLV-based vectors; (ii) vectors containing the transgene cassette directly downstream of the envelope gene are genomically more stable than those containing it within the 3′-long terminal repeat U3 region; and (iii) the genomic stability of both strains seems to be cell line dependent.
Background: Research is an increasingly important aspect of higher medical training for many doctors. Studies investigating sources of stress, isolation, and workplace bullying have not previously sought information in this setting.
Methods: An internet based questionnaire survey of doctors undertaking research (n = 259) was conducted to examine stressors and levels of job satisfaction in this potentially vulnerable group. In order to assess overall levels of satisfaction, we asked whether doctors would recommend their research post to a colleague.
Results: There was a statistically significant association between those who would not recommend their post to a colleague and those who had difficulties in arranging funding and in writing up (p<0.001). Further significant correlations were found between dissatisfaction with the post and lack of help, support, and advice from supervisors and colleagues, wanting to change supervisors, experience of the major categories of workplace bullying, and having an inadequate clinical commitment (p<0.001). When the significant variables were entered into a multivariate analysis, the results showed that dissatisfaction was associated with wanting to change supervisors and with a threat to professional status.
Conclusions: Stress and bullying are common in doctors undertaking research. These findings have important implications for medical training and for doctors choosing research projects. Setting up systems of support may have important benefits.
Replication-competent retrovirus vectors based on murine leukemia virus (MLV) have been shown to effectively transfer therapeutic genes over multiple serial infections in cell culture and through solid tumors in vivo with a high degree of genomic stability. While simple retroviruses possess a natural tumor selectivity in that they can transduce only actively dividing cells, additional tumor-targeting strategies would nevertheless be advantageous, since tumor cells are not the only actively dividing cells. In this study, we used the promiscuous murine cytomegalovirus promoter, a chimeric regulatory sequence consisting of the hepatitis B virus enhancer II and the human α1-antitrypsin (EII-Pa1AT) promoter, and a synthetic regulatory sequence consisting of a series of T-cell factor binding sites named the CTP4 promoter to generate replicating MLV vectors, whereby the last two are transcriptionally restricted to liver- and β-catenin/T-cell factor-deregulated cells, respectively. When the heterologous promoters were used to replace almost the entire MLV U3 region, including the MLV TATA box, vector replication was inefficient since nascent virus particle production from infected cells was greatly decreased. Fusion of the heterologous promoters lacking the TATA box to the MLV TATA box, however, generated vectors which replicated with almost-wild-type kinetics throughout permissive cells while exhibiting low or negligible spread in nonpermissive cells. The genomic stability of the vectors was shown to be comparable to that of a similar vector containing wild-type MLV long terminal repeats, and tropism analysis over repeated infection cycles showed that the targeted vectors retained their original specificity.
Pneumocystis carinii pneumonia (PCP) remains a serious opportunistic infection in HIV infected individuals. Seasonal changes in climate are associated with changes within individual susceptibility to infection. The possibility of monthly variability in the incidence of PCP was therefore examined by means of a cohort study of a database of 8640 HIV infected individuals attending the Chelsea and Westminster Hospital. There were 792 cases of PCP diagnosed since 1985. A marked decline was observed in the incidence of PCP in mid-1992 coincident with the introduction of PCP prophylaxis. There was a further decline in 1996 after the introduction of highly active antiretroviral therapy. Despite no significant monthly variation in the mean attendance to clinic and CD4 count, both new and all cases of PCP were higher in January than in other months (15.9% and 14.5% of all cases, respectively). A correlation with low rainfall in January and new cases of PCP was observed. These data are consistent with an influence of climatic conditions on the presentation of PCP. The diagnosis of PCP is more common in winter months suggesting that this is a transmissible infection.
SETTING—Inpatient medical wards, Department of Medicine, University Teaching Hospital, Lusaka, Zambia.
OBJECTIVE—To define the natural history, clinical presentation, and management outcome of microbiologically confirmed cryptococcal meningitis in adult AIDS patients treated under local conditions where antifungal and antiretroviral therapies are not routinely available.
DESIGN—A descriptive, longitudinal, observational study.
METHODS—All adult patients admitted to the medical wards of the University Teaching Hospital, Lusaka, Zambia with cerebrospinal fluid culture proved, primary cryptococcal meningitis, during a 12 month period were enrolled into the study. The following details were acquired: clinical features, HIV status, laboratory data, treatment accorded, and survival.
RESULTS—A total of 230 patients with primary cryptococcal meningitis were studied (median age 32 years; range 15-65 years; 112 males, 118 females). Cryptococcal meningitis was the first AIDS defining illness in 210 (91%) patients. One hundred and thirty of the 230 (56%) patients had received treatment with fluconazole monotherapy and 100 (43%) patients received palliative care only without any antifungal therapy. A 100% case fatality rate was observed in both groups at follow up: by seven weeks in the untreated group and at six months in the fluconazole treated group. The cumulative median survival from time of diagnosis was 19 days (range 1-164 days) for the fluconazole treated group and 10 days (range 0-42 days) for the untreated group.
CONCLUSION—Cryptococcal meningitis, under current treatment accorded at the University Teaching Hospital, Lusaka, has a 100% mortality in young Zambian adults with AIDS. The current treatment accorded to Zambian adults with cryptococcal meningitis is inappropriate. An urgent need exists to improve strategies for the clinical management of AIDS patients in poor African countries. The wider ethical and operational issues of making available antifungals to African AIDS patients are discussed.
Keywords: cryptococcal meningitis; Africa; AIDS/HIV; natural history