Role of interferon in Semliki Forest virus encephalitis

The type I interferon (IFN) system is a potent anti-viral innate immune response. It is primed by IFN-p and IFN-0C4, which are the immediately expressed IFNs following detection of virus infection. IFN-P establishes the anti-viral immune response within the infected cell, augments further IFN production through the induced expression of IRF-7, a transcription factor for other IFN-as, and promotes the adaptive immune response. Induction of IFN-P requires the activation of multiple transcription factors, including NF-kB; some of these are maintained in an inactive state within the cytoplasm of the resting cell. PKR is an IFN-induced, dsRNA-activated kinase capable of phosphorylating and activating the IkK, which ultimately releases NF-kB enabling its nuclear translocation. Within the nucleus NF-kB associates with IRF-3 and AP-1 on the IFN-P promoter to induce IFN-P expression. Delineation of the pathways that result in IFN-P expression has revealed viral proteins which target components of these signalling networks. To date no anti-IFN mechanisms have been observed for Semliki Forest virus (SFV), an alphavirus of the Togaviridae. The SFV genome is 11 kb in length and encodes two open reading frames; the non-structural proteins (nsP 1-4), which encode the replicase complex and the structural proteins. Studies with Sindbis virus, a closely related alphavirus have suggested that nsP2 may play a role in IFN suppression. Previous studies with SFV nsP2 observed that 50 % of nsP2 was translocated to the nucleus. When nsP2 nuclear translocation is prevented, the infection has reduced neuropathology.`This thesis explores the importance of IFN in SFV encephalitis. A quantitative PCR assay for IFN-P and IFN-a transcripts and a quantitative IFN bioassay were developed to determine differences in IFN expression under different infection conditions. Mouse models and primary cell lines were used to establish the importance of PKR for IFN-P expression during SFV infection and to determine whether SFV nsP2 has a role in modulating IFN responses. In the absence of PKR, at early times post-infection, cultured cells reproducibly produced significantly lower levels of IFN-P transcripts. Reduced levels of functional IFN were also demonstrated xii by bioassay. Previous data has shown that PKR is not required for IFN-P induction. The sensitivity of the qPCR assay has allowed the demonstration that PKR, although not critical for IFN induction, is involved in IFN-P induction and is particularly important at early time points post-infection.SFV-nsP2 has been postulated to be involved in IFN interference. Comparing SFV4 to SFV4-nsP2-RDR (a mutant virus with a single amino acid change within the nuclear localisation signal of nsP2, which prevents its translocation into the nucleus) demonstrated that relative to the number of infected cells, the SFV4nsP2-RDR mutant induced over ten-fold more IFN-P transcripts than the wildtype SFV4 strain; this upregulation was specific to IFN-p. The IFN bioassay results supported this data; SFV4-nsP2-RDR induced higher functional IFN levels in comparison to wt SFV4. Both viruses grew to similar titres and at similar rates. In the mutant and wt infections both NF-kB and IRF-3 translocated into the nucleus; however, preliminary EMSA data has suggested that the amount ofNF-kB bound to the IFN-P promoter is reduced during a wt infection. This suggests a possible mechanism for the differential IFN expression and represents the first IFN evasion mechanism described for Semliki Forest virus

PanStop: a decade of rapid containment exercises for pandemic preparedness in the WHO Western Pacific Region.

Member States in the Western Pacific Region (WPR) of the World Health Organization (WHO), are highly vulnerable to emerging influenza viruses that have the potential to cause global pandemics. A well-coordinated strategy for preparedness is required at national and international levels for a robust response and to mitigate the potentially dire consequences of the next influenza pandemic. Of 37 countries and areas in the region, 23 have publically available national pandemic influenza preparedness and response plans; of these, only 17% have been updated in the last five years. However, many plans are pending update and may benefit from integrating some components of the 2017 WHO pandemic influenza risk management guide. Additionally, many plans need to be operationalized to test their functionality. Since 2007, WPRO has conducted annual PanStop exercises to test pandemic containment plans and RC mechanisms in a simulated environment that imitates pandemic events as they unfold. PanStop exercises provide an opportunity for countries to validate policies, protocols and procedures involved in implementing rapid containment (RC) operations, and to identify strengths and opportunities for improvement in planning activities for pandemic influenza. By participating in exercises such as PanStop and the annual regional International Health Regulation (IHR) Crystal Exercise, and subsequently adapting national preparedness plans based on exercise outcomes, WPR continues to improve readiness for the next influenza pandemic.</jats:p

Semliki Forest virus nonstructural protein 2 is involved in suppression of the type I interferon response

The type I interferons (IFNs) are potent mediators of antiviral immunity, and many viruses have developed means to block their expression or their effects. Semliki Forest virus (SFV) infection induces rapid and profound silencing of host cell gene expression, a process believed to be important for the inhibition of the IFN response. In SFV-infected cells, a large proportion of the nonstructural protein nsp2 is found in the nucleus, but a role for this localization has not been described. In this work we demonstrate that a viral mutant, SFV4-RDR, in which the nuclear localization sequence of nsp2 has been rendered inactive, induces a significantly more robust IFN response in infected cells. This mutant virus replicates at a rate similar to that of the parental SFV4 strain and also shuts off host cell gene expression to similar levels, indicating that the general cellular shutoff is not responsible for the inhibition of IFN expression. Further, the rate of virus-induced nuclear translocation of early IFN transcription factors was not found to differ between the wild-type and mutant viruses, indicating that the effect of nsp2 is at a later stage. These results provide novel information about the mode of action of this viral IFN antagonist

Matrix M H5N1 vaccine induces cross-H5 clade humoral immune responses in a randomized clinical trial and provides protection from highly pathogenic influenza challenge in ferrets

Background and Methods: Highly pathogenic avian influenza (HPAI) viruses constitute a pandemic threat and the development of effective vaccines is a global priority. Sixty adults were recruited into a randomized clinical trial and were intramuscularly immunized with two virosomal vaccine H5N1 (NIBRG-14) doses (21 days apart) of 30μg HA alone or 1.5, 7.5 or 30μg HA adjuvanted with Matrix M. The kinetics and longevity of the serological responses against NIBRG-14 were determined by haemagglutination inhibition (HI), single radial haemolysis (SRH), microneutralization (MN) and ELISA assays. The cross-H5 clade responses in sera were determined by HI and the antibody-secreting (ASC) cell ELISPOT assays. The protective efficacy of the vaccine against homologous HPAI challenge was evaluated in ferrets. Results: The serological responses against the homologous and cross-reactive strains generally peaked one week after the second dose, and formulation with Matrix M augmented the responses. The NIBRG-14-specific seroprotection rates fell significantly by six months and were low against cross-reactive strains although the adjuvant appeared to prolong the longevity of the protective responses in some subjects. By 12 months post-vaccination, nearly all vaccinees had NIBRG-14-specific antibody titres below the protective thresholds. The Matrix M adjuvant was shown to greatly improve ASC and serum IgG responses following vaccination. In a HPAI ferret challenge model, the vaccine protected the animals from febrile responses, severe weight loss and local and systemic spread of the virus. Conclusion: Our findings show that the Matrix M-adjuvanted virosomal H5N1 vaccine is a promising pre-pandemic vaccine candidate

T-helper 1 cells elicited by H5N1 vaccination predict seroprotection

Vaccination is the best measure to protect the population against a potential influenza H5N1 pandemic, but 2 doses of vaccine are needed to elicit protective immune responses. An immunological marker for H5N1 vaccine effectiveness is needed for early identification of the best vaccine candidate. We conducted a phase I clinical trial of a virosomal H5N1 vaccine adjuvanted with Matrix M. Sixty adult volunteers were vaccinated intramuscularly with 2 doses of either 30 μg hemagglutinin (HA) alone or with 1.5, 7.5, or 30 μg HA and Matrix M adjuvant (50 μg). The humoral response was measured by the hemagglutination inhibition (HI), microneutralization (MN), and single radial hemolysis (SRH) assays, and the CD4(+) T-helper 1 (Th1)-cell response was measured by intracellular staining for the cytokines interleukin 2, interferon γ, and tumor necrosis factor α. The adjuvanted vaccine effectively induced CD4(+) Th1-cell responses, and the frequency of influenza-specific Th1 cells after the first vaccine dose predicted subsequent HI, MN, and SRH seroprotective responses after the second vaccination. These results support early identification of Th1-cell responses as a predictive biomarker for an efficient vaccine response, which could have great implications for early identification of persons with low or no response to vaccine when evaluating future pandemic influenza vaccine

Meningococcal carriage among a university student population-United States, 2015

Objectives : Several outbreaks of serogroup B meningococcal disease have occurred among university students in recent years. In the setting of high coverage of the quadrivalent meningococcal conjugate vaccine and prior to widespread use of serogroup B meningococcal vaccines among adolescents, we conducted surveys to characterize the prevalence and molecular characteristics of meningococcal carriage among university students.Methods: Two cross-sectional oropharyngeal carriage surveys were conducted among undergraduates at a Rhode Island university. Isolates were characterized using slide agglutination, real-time polymerase chain reaction (rt-PCR), and whole genome sequencing. Adjusted prevalence ratios and 95% confidence intervals were calculated using Poisson regression to determine risk factors for carriage.Results : A total of 1837 oropharyngeal specimens were obtained from 1478 unique participants. Overall carriage prevalence was 12.7-14.6% during the two survey rounds, with 1.8-2.6% for capsular genotype B, 0.9-1.0% for capsular genotypes C, W, or Y, and 9.9-10.8% for nongroupable strains by rt-PCR. Meningococcal carriage was associated with being male, smoking, party or club attendance, recent antibiotic use (inverse correlation), and recent respiratory infections.Conclusions : In this university setting, the majority of meningococcal carriage was due to nongroupable strains, followed by serogroup B. Further evaluation is needed to understand the dynamics of serogroup B carriage and disease among university students

The kinetics and long-term single radial haemolysis antibody response induced after vaccination.

<p>The kinetics (A) and long-term (B) single radial haemolysis response to the homologous vaccine strain A/Vietnam/1194/2004 (NIBRG-14) after vaccination with two doses (21 ±1 days apart) of inactivated virosomal H5N1 vaccine alone (30μg HA, blue) or 1.5 (red), 7.5 (green) or 30 μg HA (black) adjuvanted with Matrix-M (50μg). The sampling day after vaccination is shown on the x-axis. Each symbol represents the geometric mean lysis zone area (mm²) for one individual participant, with the group geometric mean and 95% confidence interval presented. The dotted line shows the seroprotective SRH Zone area of ≥25mm².</p

The antibody secreting cell response induced after influenza vaccination.

<p>Lymphocytes were collected 7 days after the first (day 7) and second vaccine dose (day 28) of inactivated virosomal H5N1 vaccine alone or formulated 1.5, 7.5 or 30μg HA formulated with Matrix-M (50μg). The influenza-specific IgG, IgA and IgM antibody secreting cells (ASC) were enumerated using the ELISPOT assay. The data are presented as the mean number of influenza-specific ASC per 200 000 lymphocytes ± standard error of the mean to the homologous vaccine strain A/Vietnam/1104/2004 (NIBRG-14), and the heterologous responses to A/Turkey/Turkey/1/05 (NIBRG-23), A/Cambodia/R0405050/2007 (NIBRG-88) and A/Anhui/1/05 (RG6). Statistical differences between the adjuvanted and non-adjuvanted groups were calculated by ANOVA with Dunnett’s multiple comparisons test. *p<0.05.</p

The longevity of the haemagglutination inhibition antibody response induced after vaccination.

<p>A: The haemagglutination inhibition (HI) response against the homologous vaccine strain A/Vietnam/1194/2004 (NIBRG-14) at 180 and 365 days after vaccination. B: The cross-H5 clade HI response to A/turkey/Turkey/1/2005 (NIBRG-23), A/Indonesia/5/2005 (RG2) and A/Cambodia/R0405050/2007 (NIBRG-88) at 180 days post-vaccination. Subjects were vaccinated with two doses (21 ±1 days apart) of inactivated virosomal H5N1 vaccine alone (30μg HA, blue) or 1.5 (red), 7.5 (green) or 30 μg HA (black) adjuvanted with Matrix-M (50μg). The HI titres were measured by the modified HI test using horse erythrocytes. Each symbol represents the geometric HI titre for one individual participant, with geometric mean titres for the group and 95% confidence interval presented. The dotted line shows the protective HI titre of 32.</p