Safety and response to influenza vaccine in patients with systemic-onset juvenile idiopathic arthritis receiving tocilizumab

Objective

We investigated the safety and efficacy of administering influenza vaccines to patients with systemic-onset juvenile idiopathic arthritis (sJIA) treated with tocilizumab.

Patients and methods

The subjects were 27 sJIA patients treated with tocilizumab and 17 healthy age- and sex-matched volunteers. Serum samples were collected prior to and 4–7 weeks after vaccination. Hemagglutination inhibition values of the vaccine were taken as the antibody titers. The duration of tocilizumab administration and the daily doses of prednisolone per unit body weight were analyzed to identify factors affecting the responses of the sJIA patients to influenza vaccination. We questioned all the subjects about whether they had contracted influenza and whether they had had adverse reactions to the influenza vaccination. We compared steroid doses in sJIA patients before and after vaccination to document any worsening of the underlying disease.

Results

The efficacy of influenza vaccination did not differ significantly between the sJIA group and the healthy controls. The duration of tocilizumab administration did not affect the response of the sJIA patients to the influenza vaccination. None of the sJIA patients experienced either severe adverse reactions or disease exacerbation after the influenza vaccination.

Conclusion

We found that sJIA patients treated with tocilizumab could be effectively and safely immunized with the influenza vaccine.     

Genetic characterization of seasonal influenza A (H3N2) viruses in Ontario during 2010–2011 influenza season: high prevalence of mutations at antigenic sites

Background

The direct effect of antigenic site mutations in influenza viruses on antigenic drift and vaccine effectiveness is poorly understood.

Objective

To investigate the genetic and antigenic characteristics of human influenza A (H3N2) viruses circulating in Ontario during the early 2010–2011 winter season.

Study design

We sequenced the hemagglutinin (HA) and neuraminidase (NA) genes from 41 A(H3N2) viruses detected in nasopharyngeal specimens. Strain typing was performed by hemagglutination inhibition (HI) assay. Molecular and phylogenetic tree analyses were conducted.

Results

HA and NA genes showed high similarity to the 2010–2011 vaccine strain, A/Perth/16/2009 (H3N2)-like virus (97·7–98·5% and 98·7–99·5% amino acid (AA) identity, respectively). Compared to A/Perth/16/2009 strain, HA gene mutations were documented at 28 different AA positions across all five H3 antigenic sites, with a range of 5–11 mutations in individual viruses. Thirty-six (88%) viruses had 8 AA substitutions in common; none of these had reduced HI titer. Among Ontario isolates, 11 antigenic site AAs were positively selected with an increase in glycosylation sites.

Conclusion

The presence of antigenic site mutations with high frequency among 2010–2011 influenza H3N2 isolates confirms ongoing adaptive H3N2 evolution. These may represent early phylogenetic changes that could cause antigenic drift with further mutations. Clinical relevance of antigenic site mutations not causing drift in HI assays is unknown and requires further investigation. In addition, viral sequencing information will assist with vaccine strain planning and may facilitate early detection of vaccine escape.     

A promoter mutation in the haemagglutinin segment of influenza A virus generates an effective candidate live attenuated vaccine

Background

Annual seasonal and pandemic influenza vaccines need to be produced in a very tight time frame. Haemagglutinin (HA) is the major immunogenic component of influenza vaccines, and there is a lot of interest in improving candidate vaccine viruses.

Objectives

It has been shown elsewhere that mutations introduced in the non-coding region of influenza genome segments can upregulate protein expression. Our objective was to assess a virus based on the laboratory strain A/PR/8/34 (PR8) containing a modified 3′ non-coding region of RNA segment 4 (haemagglutinin).

Methods

NIBRG-93 was generated using reverse genetics. HA protein expression and growth properties were assessed. The virus phenotype suggested that it could be a candidate for use as a live attenuated vaccine, so in vivo studies were performed to assess its suitability.

Results

NIBRG-93 virus has enhanced haemagglutinin production and is significantly attenuated. Electron microscopy (EM) shows that the modified virus produces a large proportion of ‘virus-like particles’ that consist of budded cell membrane covered in HA but lacking M1 protein. The virus was shown to be attenuated in mice and offered complete protection against lethal challenge.

Conclusions

We demonstrate that NIBRG-93 is an effective live attenuated vaccine virus protecting mice against lethal challenge and reducing virus shedding.     

Vaccination of monoglycosylated hemagglutinin induces cross-strain protection against influenza virus infections

The 2009 H1N1 pandemic and recent human cases of H5N1, H7N9, and H6N1 in Asia highlight the need for a universal influenza vaccine that can provide cross-strain or even cross-subtype protection. Here, we show that recombinant monoglycosylated hemagglutinin (HA_mg) with an intact protein structure from either seasonal or pandemic H1N1 can be used as a vaccine for cross-strain protection against various H1N1 viruses in circulation from 1933 to 2009 in mice and ferrets. In the HA_mg vaccine, highly conserved sequences that were originally covered by glycans in the fully glycosylated HA (HA_fg) are exposed and thus, are better engulfed by dendritic cells (DCs), stimulated better DC maturation, and induced more CD8+ memory T cells and IgG-secreting plasma cells. Single B-cell RT-PCR followed by sequence analysis revealed that the HA_mg vaccine activated more diverse B-cell repertoires than the HA_fg vaccine and produced antibodies with cross-strain binding ability. In summary, the HA_mg vaccine elicits cross-strain immune responses that may mitigate the current need for yearly reformulation of strain-specific inactivated vaccines. This strategy may also map a new direction for universal vaccine design.HA glycoprotein on the surface of influenza virus is a major target for infectivity-neutralizing antibodies. However, the antigenic drift and shift of this protein mean that influenza vaccines must be reformulated annually to include HA proteins of the viral strains predicted for the upcoming flu season (1). This time-consuming annual reconfiguration process has led to efforts to develop new strategies and identify conserved epitopes recognized by broadly neutralizing antibodies as the basis for designing universal vaccines to elicit antibodies with a broad protection against various strains of influenza infection (2–6). Previous studies have shown that the stem region of HA is more conserved and able to induce cross-reactive and broadly neutralizing antibodies (7–9) to prevent the critical fusion of viral and endosomal membranes in the influenza lifecycle (10–14). Other broadly neutralizing antibodies have been found to bind regions near the receptor binding site of the globular domain, although these antibodies are fewer in number (15, 16).Posttranslational glycosylation of HA plays an important role in the lifecycle of the influenza virus and also contributes to the structural integrity of HA and the poor immune response of the infected hosts. Previously, we trimmed down the size of glycans on avian influenza H5N1 HA with enzymes and showed that H5N1 HA with a single N-linked GlcNAc at each glycosylation site [monoglycosylated HA (HA_mg)] produces a superior vaccine with more enhanced antibody response and neutralization activity against the homologous influenza virus than the fully glycosylated HA (HA_fg) (17). Here, to test whether the removal of glycans from HA contributes to better immune responses and possibly protects against heterologous strains of influenza viruses, we compared and evaluated the efficacy of HA glycoproteins with various lengths of glycans as potential vaccine candidates.     

A recombinant herpes virus expressing influenza hemagglutinin confers protection and induces antibody-dependent cellular cytotoxicity

Despite widespread yearly vaccination, influenza leads to significant morbidity and mortality across the globe. To make a more broadly protective influenza vaccine, it may be necessary to elicit antibodies that can activate effector functions in immune cells, such as antibody-dependent cellular cytotoxicity (ADCC). There is growing evidence supporting the necessity for ADCC in protection against influenza and herpes simplex virus (HSV), among other infectious diseases. An HSV-2 strain lacking the essential glycoprotein D (gD), was used to create ΔgD-2, which is a highly protective vaccine against lethal HSV-1 and HSV-2 infection in mice. It also elicits high levels of IgG2c antibodies that bind FcγRIV, a receptor that activates ADCC. To make an ADCC-eliciting influenza vaccine, we cloned the hemagglutinin (HA) gene from an H1N1 influenza A strain into the ΔgD-2 HSV vector. Vaccination with ΔgD-2::HA_PR8 was protective against homologous influenza challenge and elicited an antibody response against HA that inhibits hemagglutination (HAI⁺), is predominantly IgG2c, strongly activates FcγRIV, and protects against influenza challenge following passive immunization of naïve mice. Prior exposure of mice to HSV-1, HSV-2, or a replication-defective HSV-2 vaccine (dl5-29) does not reduce protection against influenza by ΔgD-2::HA_PR8. This vaccine also continues to elicit protection against both HSV-1 and HSV-2, including high levels of IgG2c antibodies against HSV-2. Mice lacking the interferon-α/β receptor and mice lacking the interferon-γ receptor were also protected against influenza challenge by ΔgD-2::HA_PR8. Our results suggest that ΔgD-2 can be used as a vaccine vector against other pathogens, while also eliciting protective anti-HSV immunity.

Influenza remains a global health threat. Seasonal strains of influenza A and B cause an estimated 5 million cases of severe infections and 500,000 deaths per year (1). Influenza pandemics have caused even greater morbidity and mortality. During the H1N1 pandemic of 1918 to 1919, 500 million people, approximately one-third of the world’s population at that time, were estimated to have been infected with this strain, leading to 50 million deaths (2). The H1N1 pandemic of 2009 is estimated to have caused up to 575,000 deaths (2). Currently, three types of influenza vaccines are offered annually in the United States: a recombinant virus expressing influenza proteins, chemically inactivated virus, and live attenuated virus (3). Regardless of the vaccine type, multiple strains are included to increase the chances of developing sufficient protection against major circulating influenza strains. However, these vaccines primarily elicit a neutralizing antibody response that is sensitive to changes in the influenza virus due to antigenic drift and shift (4). Antigenic drift results from an accumulation of random mutations in influenza antigens, like hemagglutinin (HA), altering sites recognized by the immune system (4). Influenza A strains can also undergo antigenic shift, whereby two different influenza strains infect the same cell to form a reassortant virus with new antigenic properties (4). Due to limited immunity in the population, these new strains are highly virulent, causing widespread epidemics and disease (4). With antigenic drift and shift, vaccine-mediated protection against circulating strains has been insufficient (5). Influenza vaccines that elicit more robust and long-term protection are therefore needed. Notably, if an influenza vaccine with ≥75% efficacy were to be broadly used in the United States, an estimated 19,500 deaths a year could be prevented and direct healthcare costs reduced by $3.5 billion (6).For many years, efforts to improve influenza vaccines have focused on eliciting an immune response for full, broad protection against both circulating and future strains of the virus. These studies have shown that, in general, neutralizing antibodies are sufficient for homologous protection (7). However, achieving heterologous protection may require more broadly neutralizing antibodies or nonneutralizing antibodies able to activate effector immune cells (5). Previous studies have found that passively transferred nonneutralizing monoclonal antibodies can be potently protective in a mouse influenza challenge model (8–10). Several novel strategies have attempted to generate a nonneutralizing response against influenza. For example, vaccines have been created to specifically target the conserved stem region of HA (11–13).Nonneutralizing antibodies stimulate effector cell mechanisms, including antibody-mediated phagocytosis and antibody-dependent cellular cytotoxicity (ADCC), both of which require activation of the Fcγ receptors (FcγRs) (14). Specific isotypes of IgG antibodies are associated with FcγR modulation and subsequent ADCC activation, including the IgG1 and IgG3 subtypes in humans, as well as IgG2a and IgG2c subtypes in mice (15–19). IgG2a and IgG2c isotypes are functionally equivalent and mouse strain-dependent, with IgG2c present in C57BL/6J mice (20). Recent studies have demonstrated that natural infection by influenza and vaccination elicit nonneutralizing antibodies with effector functions that contribute to protection (5, 9, 21–27). In mouse and nonhuman primate challenge models, ADCC-mediating antibodies have demonstrated protection against both homologous and heterologous influenza challenge (9, 28).Recently, we developed a single-cycle herpes simplex virus (HSV) vaccine that completely protects against vaginal, skin, and ocular challenges by HSV-1 and HSV-2 (29, 30). Protection elicited by this vaccine, designated ΔgD-2 for its lack of the essential glycoprotein D (gD) gene, is transferable via passive infusion of immune sera to naïve wild-type mice but not to mice lacking the Fcγ common chain (30). The immune response elicited by ΔgD-2 primarily elicits nonneutralizing antibodies with high levels of FcγRIV-activating function.We asked whether ΔgD-2 could be used as a vaccine platform to induce broadly protective FcγRIV-activating antibodies against a heterologous antigen, such as influenza HA. In this study, we demonstrate that our recombinant vaccine, ΔgD-2::HA_PR8, elicits protection against influenza with a high proportion of FcγRIV-activating antibodies. Additionally, anticipating the use of ΔgD-2 as a vaccine vector against other pathogens, we tested whether our construct would still be protective in mice lacking interferon (IFN) function. Many humans have inborn errors in their IFN signaling pathways, leading to more lethal outcomes in infection (31). Patients with such deficiencies are disproportionately represented among HSV encephalitis cases and are often diagnosed only after presenting with serious symptoms (32–38). This at-risk population underscores the importance of eliciting protection against HSV in the absence of a functional IFN-α/β response. Additionally, many pathogens, such as dengue virus, require mouse models lacking IFN function, and for ease of testing, an efficacious vaccine should remain functional in these mice (39–41). In this study, we demonstrate that ΔgD-2 is a versatile, immunogenic vaccine vector that provides a strong FcγRIV-activating immune response against heterologous pathogens, while maintaining its protective benefit against HSV, in both wild-type and IFN-deficient mice.     

Migratory birds in southern Brazil are a source of multiple avian influenza virus subtypes

Background

There is insufficient knowledge about the relation of avian influenza virus (AIV) to migratory birds in South America. Accordingly, we studied samples obtained over a 4‐year period (2009‐2012) from wild birds at a major wintering site in southern Brazil.

Methods

We obtained 1212 oropharyngeal/cloacal samples from wild birds at Lagoa do Peixe National Park and screened them for influenza A virus by RT‐PCR amplification of the matrix gene. Virus isolates were subjected to genomic sequencing and antigenic characterization.

Results

Forty‐eight samples of 1212 (3.96%) contained detectable influenza virus RNA. Partial viral sequences were obtained from 12 of these samples, showing the presence of H2N2 (1), H6Nx (1), H6N1 (8), H9N2 (1), and H12N5 (1) viruses. As H6 viruses predominated, we generated complete genomes from all 9 H6 viruses. Phylogenetic analyses showed that they were most similar to viruses of South American lineage. The H6N1 viruses caused no disease signs in infected ferrets and, despite genetic differences, were antigenically similar to North American isolates.

Conclusions

Lagoa do Peixe National Park is a source of multiple AIV subtypes, with the levels of influenza virus in birds being highest at the end of their wintering period in this region. H6N1 viruses were the predominant subtype identified. These viruses were more similar to viruses of South American lineage than to those of North American lineage.

     

Clinical characteristics of influenza virus infection in juvenile idiopathic arthritis patients treated with tocilizumab

Background

Inhibition of interleukin-6 (IL-6) signaling by tocilizumab is highly effective for treatment of refractory juvenile idiopathic arthritis (JIA). It appears that IL-6 plays an important role in the immune response to the influenza virus, but it is not clear whether treatment with tocilizumab affects the severity of influenza.

Methods

We retrospectively collected clinical and laboratory data from JIA patients (n = 33) treated with tocilizumab. Ten patients who developed influenza (tocilizumab group; 10.1 %, 10/99 patient-years) were analyzed. Eleven JIA patients who experienced influenza during conventional treatments, without tocilizumab (control group), were compared with the tocilizumab group.

Results

Of the 10 patients in the tocilizumab group, 6 patients did not have high fever (>38 °C), and the other 4 febrile patients recovered from fever in 1 day. White blood cell counts and lymphocyte counts were significantly lower at the acute phase of infection compared with data from before influenza infection. The degree of fever and level of C-reactive protein in the tocilizumab group were significantly reduced compared with the control group.

Conclusions

IL-6 inhibition by tocilizumab reduced inflammation associated with infection and resulted in mild symptoms during influenza. Leukopenia might be a useful indicator of viral infection, including influenza, during tocilizumab treatment.     

Optimisation of a micro‐neutralisation assay and its application in antigenic characterisation of influenza viruses

Objectives

The identification of antigenic variants and the selection of influenza viruses for vaccine production are based largely on antigenic characterisation of the haemagglutinin (HA) of circulating viruses using the haemagglutination inhibition (HI) assay. However, in addition to evolution related to escape from host immunity, variants emerging as a result of propagation in different cell substrates can complicate the interpretation of HI results. The objective was to develop further a micro-neutralisation (MN) assay to complement the HI assay in antigenic characterisation of influenza viruses to assess the emergence of new antigenic variants and reinforce the selection of vaccine viruses.

Design and setting

A 96-well-plate plaque reduction MN assay based on the measurement of infected cell population using a simple imaging technique.

Sample

Representative influenza A (H1N1) pdm09, A(H3N2) and B viruses isolated between 2004 and 2013

Main outcome measures and results

Improvements to the plaque reduction MN assay included selection of the most suitable cell line according to virus type or subtype, and optimisation of experimental design and data quantitation. Comparisons of the results of MN and HI assays showed the importance of complementary data in determining the true antigenic relationships among recent human influenza A(H1N1)pdm09, A(H3N2) and type B viruses.

Conclusions

Our study demonstrates that the improved MN assay has certain advantages over the HI assay: it is not significantly influenced by the cell-selected amino acid substitutions in the neuraminidase (NA) of A(H3N2) viruses, and it is particularly useful for antigenic characterisation of viruses which either grow to low HA titre and/or undergo an abortive infection resulting in an inability to form plaques in cultured cells.     

A common solution to group 2 influenza virus neutralization

The discovery and characterization of broadly neutralizing antibodies (bnAbs) against influenza viruses have raised hopes for the development of monoclonal antibody (mAb)-based immunotherapy and the design of universal influenza vaccines. Only one human bnAb (CR8020) specifically recognizing group 2 influenza A viruses has been previously characterized that binds to a highly conserved epitope at the base of the hemagglutinin (HA) stem and has neutralizing activity against H3, H7, and H10 viruses. Here, we report a second group 2 bnAb, CR8043, which was derived from a different germ-line gene encoding a highly divergent amino acid sequence. CR8043 has in vitro neutralizing activity against H3 and H10 viruses and protects mice against challenge with a lethal dose of H3N2 and H7N7 viruses. The crystal structure and EM reconstructions of the CR8043-H3 HA complex revealed that CR8043 binds to a site similar to the CR8020 epitope but uses an alternative angle of approach and a distinct set of interactions. The identification of another antibody against the group 2 stem epitope suggests that this conserved site of vulnerability has great potential for design of therapeutics and vaccines.Influenza viruses are a significant and persistent threat to human health worldwide. Annual epidemics cause 3–5 million cases of severe illness and up to 0.5 million deaths (1), and periodic influenza pandemics have the potential to kill millions (2). Inhibitors against the viral surface glycoprotein neuraminidase are widely used for the treatment of influenza infections, but their efficacy is being compromised by the emergence of drug-resistant viral strains (3). Vaccination remains the most effective strategy to prevent influenza virus infection. However, protective efficacy is suboptimal in the highest risk groups: infants, the elderly, and the immunocompromised (1). Furthermore, because immunity after vaccination is typically strain-specific and influenza viruses evolve rapidly, vaccines must be updated almost annually. The antigenic composition of the vaccine is based on a prediction of strains likely to circulate in the coming year, therefore, mismatches between vaccine strains and circulating strains occur that can render the vaccine less effective (4). Consequently, there is an urgent need for new prophylactic and therapeutic interventions that provide broad protection against influenza.Immunity against influenza viruses is largely mediated by neutralizing antibodies that target the major surface glycoprotein hemagglutinin (HA) (5, 6). Identification of antigenic sites on HA indicates that influenza antibodies are primarily directed against the immunodominant HA head region (7), which mediates endosomal uptake of the virus into host cells by binding to sialic acid receptors (8). Because of high mutation rates in the HA head region and its tolerance for antigenic changes, antibodies that target the HA head are typically only effective against strains closely related to the strain(s) by which they were elicited, although several receptor binding site-targeting antibodies with greater breadth have been structurally characterized (9–15). In contrast, antibodies that bind to the membrane-proximal HA stem region tend to exhibit much broader neutralizing activity and can target strains within entire subtypes and groups (16–25) as well as across influenza types (24). These stem-directed antibodies inhibit major structural rearrangements in HA that are required for the fusion of viral and host endosomal membranes and thus, prevent the release of viral contents into the cell (8). The stem region is less permissive for mutations than the head and relatively well-conserved across divergent influenza subtypes.Anti-stem antibodies are elicited in some, but not all, individuals during influenza infection or vaccination (20, 26) and thus, hold great promise as potential broad spectrum prophylactic or therapeutic agents and for the development of a universal influenza vaccine (27–29). The majority of the known heterosubtypic stem binding antibodies neutralize influenza A virus subtypes belonging to group 1 (17–20, 23, 25). Furthermore, two antibodies that target a similar epitope in the HA stem, like most heterosubtypic group 1 antibodies, are able to more broadly recognize both group 1 and 2 influenza A viruses (22) or influenza A and B viruses (24). Strikingly, group 2-specific broadly neutralizing Abs (bnAbs) seem to be rare, because only one has been reported to date (21). CR8020 uniquely targets a distinct epitope in the stem in close proximity to the viral membrane at the HA base and binds lower down the stem than any other influenza HA antibody (21).In the discovery process that led to the isolation of bnAb CR8020, we recovered additional group 2-specific bnAbs. Here, we describe one such bnAb, CR8043, which recognizes a similar but nonidentical footprint on the HA as CR8020 and approaches the HA from a different angle. Furthermore, these two bnAbs are derived from different germ-line genes and, consequently, use distinct sets of interactions for HA recognition. Thus, the human immune system is able to recognize this highly conserved epitope in different ways using different germ-line genes. Hence, this valuable information can be used for the design of therapeutics and vaccines targeting this site of vulnerability in group 2 influenza A viruses that include the pandemic H3N2 subtype.     

An analysis of national target groups for monovalent 2009 pandemic influenza vaccine and trivalent seasonal influenza vaccines in 2009-10 and 2010-11

Background

Vaccination is generally considered to be the best primary prevention measure against influenza virus infection. Many countries encourage specific target groups of people to undertake vaccination, often with financial subsidies or a priority list. To understand differential patterns of national target groups for influenza vaccination before, during and after the 2009 influenza pandemic, we reviewed and analyzed the country-specific policies in the corresponding time periods.

Methods

Information on prioritized groups targeted to receive seasonal and pandemic influenza vaccines was derived from a multi-step internet search of official health department websites, press releases, media sources and academic journal articles. We assessed the frequency and consistency of targeting 20 different groups within populations which are associated with age, underlying medical conditions, role or occupations among different countries and vaccines. Information on subsidies provided to specific target groups was also extracted.

Results

We analyzed target groups for 33 (seasonal 2009 and 2009-10 vaccines), 72 (monovalent pandemic 2009-10 vaccine) and 34 (seasonal 2010 and 2010-11 vaccines) countries. In 2009-10, the elderly, those with chronic illness and health care workers were common targets for the seasonal vaccine. Comparatively, the elderly, care home residents and workers, animal contacts and close contacts were less frequently targeted to receive the pandemic vaccine. Pregnant women, obese persons, essential community workers and health care workers, however, were more commonly targeted. After the pandemic, pregnant women, obese persons, health care and care home workers, and close contacts were more commonly targeted to receive the seasonal vaccine compared to 2009-10, showing continued influence from the pandemic. Many of the countries provided free vaccines, partial subsidies, reimbursements or national health insurance coverage to specific target groups and over one-third of the countries offered universal subsidy regarding the pandemic vaccine. There was also some inconsistency between countries in target groups.

Conclusions

Differences in target groups between countries may reflect variable objectives as well as uncertainties regarding the transmission dynamics, severity and age-specific immunity against influenza viruses before and after vaccination. Clarification on these points is essential to elucidate optimal and object-oriented vaccination strategies.

     

Multiannual forecasting of seasonal influenza dynamics reveals climatic and evolutionary drivers

Human influenza occurs annually in most temperate climatic zones of the world, with epidemics peaking in the cold winter months. Considerable debate surrounds the relative role of epidemic dynamics, viral evolution, and climatic drivers in driving year-to-year variability of outbreaks. The ultimate test of understanding is prediction; however, existing influenza models rarely forecast beyond a single year at best. Here, we use a simple epidemiological model to reveal multiannual predictability based on high-quality influenza surveillance data for Israel; the model fit is corroborated by simple metapopulation comparisons within Israel. Successful forecasts are driven by temperature, humidity, antigenic drift, and immunity loss. Essentially, influenza dynamics are a balance between large perturbations following significant antigenic jumps, interspersed with nonlinear epidemic dynamics tuned by climatic forcing.Influenza outbreaks have been documented in the scientific literature in records that extend back to at least 1650 (1), making it an exceptional example of a persisting, recurrent disease. Being a respiratory infection, influenza spreads rapidly from person to person through a population in the form of virus particles airborne as respiratory droplets or aerosols. Depending on the circumstances, influenza typically infects between 10% and 50% of a given population and has become a source of considerable human morbidity and mortality (2). There is much controversy in identifying the seasonal drivers that generate annual influenza epidemics and the processes that give rise to their large variability (3–12). This is an outstanding problem of influenza research today. Using long-term modeling, a recent study (9) gave support to the possibility that absolute humidity is the predominant determinant of influenza seasonality in temperate zones, driving disease transmission and controlling the timing of individual wintertime outbreaks. Another study investigated the physical properties of absolute humidity on influenza virus transmission and influenza virus survival (3). However, a general understanding of the mechanisms underlying influenza seasonal variation remains quite limited (8). Here, we use a simple mathematical model to unravel the interplay between climate and evolution to predict long-term influenza dynamics correctly for the years since June 2010.A requirement for the generation of recurrent epidemics is a sufficient and continuous source of new susceptible individuals arising in the population, enough to fuel each new outbreak (13). In the case of influenza, infected individuals recover with immunity but eventually become susceptible again because of the rapidly evolving nature of the influenza virus (7, 14). Positive selection exerted by the host immune system leads to a continual antigenic drift of the influenza virus’s glycoproteins, particularly the main antigen, hemagglutinin, thus allowing the virus to eventually evade the immune system (15). The process of antigenic drift thereby creates an important renewed source of susceptible individuals. Hence, evolutionary forces are considered tremendously important in shaping complex recurrent patterns of infectious diseases and explain why influenza is regarded as “an invariable disease caused by a variable virus” (1).The changing rate of antigenic drift also has a significant impact on the timing and amplitude of influenza outbreaks (16). Recent studies reveal that the evolution of influenza A H3N2’s main antigen is punctuated in character such that the drift occurs within discrete antigenic clusters (neutral periods), but with jumps to newly arising clusters after irregular periods (17, 18). A significant jump for the A H3N2 lineage last occurred during the 2003–4 season with the appearance of the A/Fujian virus strain coinciding with a sharp influenza outbreak approximately 2 mo earlier than usual, with a normal attack rate. Nevertheless, it is difficult to demonstrate a consistent and conclusive direct link between the size of antigenic jumps and changes in influenza dynamics at the population level (6, 19–21).     

IL-15 adjuvanted multivalent vaccinia-based universal influenza vaccine requires CD4+ T cells for heterosubtypic protection

Current influenza vaccines are ineffective against novel viruses and the source or the strain of the next outbreak of influenza is unpredictable; therefore, establishing universal immunity by vaccination to limit the impact of influenza remains a high priority. To meet this challenge, a novel vaccine has been developed using the immunogenic live vaccinia virus as a vaccine vector, expressing multiple H5N1 viral proteins (HA, NA, M1, M2, and NP) together with IL-15 as a molecular adjuvant. Previously, this vaccine demonstrated robust sterile cross-clade protection in mice against H5 influenza viruses, and herein its use has been extended to mediate heterosubtypic immunity toward viruses from both group 1 and 2 HA lineages. The vaccine protected mice against lethal challenge by increasing survival and significantly reducing lung viral loads against the most recent human H7N9, seasonal H3N2, pandemic-2009 H1N1, and highly pathogenic H7N7 influenza A viruses. Influenza-specific antibodies elicited by the vaccine failed to neutralize heterologous viruses and were unable to confer protection by passive transfer. Importantly, heterologous influenza-specific CD4⁺ and CD8⁺ T-cell responses that were elicited by the vaccine were effectively recalled and amplified following viral challenge in the lungs and periphery. Selective depletion of T-cell subsets in the immunized mice revealed an important role for CD4⁺ T cells in heterosubtypic protection, despite low sequence conservation among known MHC-II restricted epitopes across different influenza viruses. This study illustrates the potential utility of our multivalent Wyeth/IL-15/5Flu as a universal influenza vaccine with a correlate of protective immunity that is independent of neutralizing antibodies.Influenza causes widespread infection during seasonal epidemics and occasional worldwide pandemics despite available vaccines. The subtype of future outbreaks or pandemic influenza strains is unpredictable as is its source, evident from the most recent H7N9 outbreak from poultry in China, the variant H3N2 outbreak from swine in the United States in 2012, the H1N1 worldwide pandemic of 2009, and the highly pathogenic avian influenza (HPAI) H5N1 in 1997 from domestic poultry. Therefore, the development of a successful universal vaccination strategy is urgently needed.Universal protection requires heterosubtypic immunity (HSI), whereby vaccination against one influenza virus cross-protects against novel and emerging strains that could potentially be mediated by multiple adaptive immune mechanisms. T cells are potent mediators of HSI, because these cells typically recognize peptide epitopes derived from internal proteins of influenza virus, which are naturally more conserved than surface HA and NA across different strains and even serologically distinct viral subtypes. Because of sequence conservation of the majority of T-cell epitopes between different influenza viruses, cross-reactive T-cell responses have been detected in healthy seronegative individuals against H5N1 and pandemic H1N1-2009 viruses (1, 2).Previously, we generated a multivalent vaccinia virus-based H5N1 influenza vaccine, which demonstrated effective cross-clade immunity against lethal H5N1 challenges. This vaccine expresses five H5N1-derived influenza proteins (HA, NA, M1, M2, and NP), in combination with the immune stimulatory cytokine IL-15 (3) that increases long-term memory responses, along with enhanced T-cell, B-cell, and NK cell functions, including cytokine production and survival. Despite the use of live vaccine vectors being generally constrained in immune-compromised individuals, the vaccine vector (Wyeth/IL-15) has been proven safe and effective in primates and immune-deficient mice (4).A cell-culture–derived, live vaccine vector that encodes full-length influenza proteins with inherent capacity to access MHC-I and II processing pathways to establish robust influenza-specific T-cell responses is an excellent approach to overcome issues related to population-wide MHC polymorphism and egg-based production methods for HPAI vaccines. Herein, we extend the use of Wyeth/IL-15/5Flu vaccine against multiple human influenza viruses of different HA subtypes, including the highly pathogenic H7N7, pandemic H1N1-2009, seasonal H3N2, and the most recent human H7N9 viruses. The vaccine proved effective against all of the heterologous strains tested, and the immunological mechanisms of protection were investigated to decipher correlates of immunity.     

A monoclonal antibody‐based immunoassay for measuring the potency of 2009 pandemic influenza H1N1 vaccines

Background

The potency of inactivated influenza vaccines is determined using a single radial immunodiffusion (SRID) assay. This assay is relatively easy to standardize, it is not technically demanding, and it is capable of measuring the potency of several vaccine strain subtypes in a multivalent vaccine. Nevertheless, alternative methods that retain the major advantages of the SRID, but with a greater dynamic range of measurement and with reduced reagent requirements, are needed.

Objectives

The feasibility of an ELISA-based assay format was explored as an alternative potency assay for inactivated influenza vaccines.

Methods

Several murine monoclonal antibodies (mAbs), specific for the 2009 pandemic H1N1 influenza virus hemagglutinin (HA), were evaluated for their potential to capture and quantify HA antigen. Vaccine samples, obtained from four licensed influenza vaccine manufacturers, included monovalent bulk vaccine, monovalent vaccine, and trivalent vaccine. Traditional SRID potency assays were run in parallel with the mAb–ELISA potency assay using the reference antigen standard appropriate for the vaccine samples being tested.

Results

The results indicated that the ELISA potency assay can quantify HA over a wide range of concentrations, including vaccine at subpotent doses, and the ELISA and SRID potency values correlated well for most vaccine samples. Importantly, the assay was capable of quantifying A/California HA in a trivalent formulation.

Conclusions

This study demonstrates the general feasibility of the mAb approach and strongly suggests that such ELISAs have potential for continued development as an alternative method to assay the potency of inactivated influenza vaccines.     

The role of facemasks and hand hygiene in the prevention of influenza transmission in households: results from a cluster randomised trial; Berlin, Germany, 2009-2011

Background

Previous controlled studies on the effect of non-pharmaceutical interventions (NPI) - namely the use of facemasks and intensified hand hygiene - in preventing household transmission of influenza have not produced definitive results. We aimed to investigate efficacy, acceptability, and tolerability of NPI in households with influenza index patients.

Methods

We conducted a cluster randomized controlled trial during the pandemic season 2009/10 and the ensuing influenza season 2010/11. We included households with an influenza positive index case in the absence of further respiratory illness within the preceding 14 days. Study arms were wearing a facemask and practicing intensified hand hygiene (MH group), wearing facemasks only (M group) and none of the two (control group). Main outcome measure was laboratory confirmed influenza infection in a household contact. We used daily questionnaires to examine adherence and tolerability of the interventions.

Results

We recruited 84 households (30 control, 26 M and 28 MH households) with 82, 69 and 67 household contacts, respectively. In 2009/10 all 41 index cases had a influenza A (H1N1) pdm09 infection, in 2010/11 24 had an A (H1N1) pdm09 and 20 had a B infection. The total secondary attack rate was 16% (35/218). In intention-to-treat analysis there was no statistically significant effect of the M and MH interventions on secondary infections. When analysing only households where intervention was implemented within 36 h after symptom onset of the index case, secondary infection in the pooled M and MH groups was significantly lower compared to the control group (adjusted odds ratio 0.16, 95% CI, 0.03-0.92). In a per-protocol analysis odds ratios were significantly reduced among participants of the M group (adjusted odds ratio, 0.30, 95% CI, 0.10-0.94). With the exception of MH index cases in 2010/11 adherence was good for adults and children, contacts and index cases.

Conclusions

Results suggest that household transmission of influenza can be reduced by the use of NPI, such as facemasks and intensified hand hygiene, when implemented early and used diligently. Concerns about acceptability and tolerability of the interventions should not be a reason against their recommendation.

Trial registration

The study was registered with ClinicalTrials.gov (Identifier NCT00833885).     

Whole genome sequencing identifies influenza A H3N2 transmission and offers superior resolution to classical typing methods

Objectives

Influenza with its annual epidemic waves is a major cause of morbidity and mortality worldwide. However, only little whole genome data are available regarding the molecular epidemiology promoting our understanding of viral spread in human populations.

Methods

We implemented a RT-PCR strategy starting from patient material to generate influenza A whole genome sequences for molecular epidemiological surveillance. Samples were obtained within the Bavarian Influenza Sentinel. The complete influenza virus genome was amplified by a one-tube multiplex RT-PCR and sequenced on an Illumina MiSeq.

Results

We report whole genomic sequences for 50 influenza A H3N2 viruses, which was the predominating virus in the season 2014/15, directly from patient specimens. The dataset included random samples from Bavaria (Germany) throughout the influenza season and samples from three suspected transmission clusters. We identified the outbreak samples based on sequence identity. Whole genome sequencing (WGS) was superior in resolution compared to analysis of single segments or partial segment analysis. Additionally, we detected manifestation of substantial amounts of viral quasispecies in several patients, carrying mutations varying from the dominant virus in each patient.

Conclusion

Our rapid whole genome sequencing approach for influenza A virus shows that WGS can effectively be used to detect and understand outbreaks in large communities. Additionally, the genomic data provide in-depth details about the circulating virus within one season.

     

Hip fracture incidence in the elderly in Austria: An epidemiological study covering the years 1994 to 2006

Background

Previous studies have shown influenza vaccine uptake in UK nursing home residents to be low. Very little information exists regarding the uptake of pneumococcal vaccine in this population. The formulation of policies relating to the vaccination of residents has been proposed as a simple step that may help improve vaccine uptake in care homes.

Methods

A postal questionnaire was sent to matrons of all care homes with nursing within the Greater Nottingham area in January 2006. Non respondents were followed up with up to 3 phone calls.

Results

30% (16/53) of respondents reported having a policy addressing influenza vaccination and 15% (8/53) had a policy addressing pneumococcal vaccination. Seasonal influenza vaccine coverage in care homes with a vaccination policy was 87% compared with 84% in care homes without a policy (p = 0.47). The uptake of pneumococcal vaccination was found to be low, particularly in care homes with no vaccination policy. Coverage was 60% and 32% in care homes with and without a vaccination policy respectively (p = 0.06). This result was found to be statistically significant on multivariate analysis (p = 0.03, R = 0.46)

Conclusion

The uptake of influenza vaccine among care home residents in the Nottingham region is relatively high, although pneumococcal vaccine uptake is low. This study shows that there is an association between pneumococcal vaccine uptake and the existence of a vaccination policy in care homes, and highlights that few care homes have vaccination policies in place.     

Variation in protection of four divergent avian influenza virus vaccine seed strains against eight clade 2.2.1 and 2.2.1.1. Egyptian H5N1 high pathogenicity variants in poultry

Background

Highly pathogenic (HP) H5N1 avian influenza virus (AIV) was introduced to Egyptian poultry in 2006 and has since become enzootic. Vaccination has been utilized as a control tool combined with other control methods, but for a variety of reasons, the disease has not been eradicated. In 2007, an antigenically divergent hemagglutinin subclade, 2.2.1.1, emerged from the original clade 2.2.1 viruses.

Objectives

The objective was to evaluate four diverse AIV isolates for use as vaccines in chickens, including two commercial vaccines and two additional contemporary isolates, against challenge with numerous clade 2.2.1 and clade 2.2.1.1 H5N1 HPAIV Egyptian isolates to assess the variation in protection among different vaccine and challenge virus combinations.

Methods

Vaccination-challenge studies with four vaccines and up to eight challenge strains with each vaccine for a total of 25 vaccination-challenge groups were conducted with chickens. An additional eight groups served as sham-vaccinated controls. Mortality, mean death time, morbidity, virus, and pre-challenge antibodies were evaluated as metrics of protection. Hemagglutination inhibition data were used to visualize the antigenic relatedness of the isolates.

Results and conclusions

Although all but one vaccine-challenge virus combination significantly reduced shed and mortality as compared to sham vaccinates, there were differences in protection among the vaccines relative to one another based on challenge virus. This emphasizes the difficulty in vaccinating against diverse, evolving virus populations, and the importance of selecting optimal vaccine seed strains for successful HPAIV control.