Sequential Influenza Research Articles

Tracking of activated cTfh cells following sequential influenza vaccinations reveals transcriptional profile of clonotypes driving a vaccine-induced immune response.

A vaccine against influenza is available seasonally but is not 100% effective. A predictor of successful seroconversion in adults is an increase in activated circulating T follicular helper (cTfh) cells after vaccination. However, the impact of repeated annual vaccinations on long-term protection and seasonal vaccine efficacy remains unclear. In this study, we examined the T cell receptor (TCR) repertoire and transcriptional profile of vaccine-induced expanded cTfh cells in individuals who received sequential seasonal influenza vaccines. We measured the magnitude of cTfh and plasmablast cell activation from day 0 (d0) to d7 post-vaccination as an indicator of a vaccine response. To assess TCR diversity and T cell expansion we sorted activated and resting cTfh cells at d0 and d7 post-vaccination and performed TCR sequencing. We also single cell sorted activated and resting cTfh cells for TCR analysis and transcriptome sequencing. The percent of activated cTfh cells significantly increased from d0 to d7 in each of the 2016-17 (p < 0.0001) and 2017-18 (p = 0.015) vaccine seasons with the magnitude of cTfh activation increase positively correlated with the frequency of circulating plasmablast cells in the 2016-17 (p = 0.0001) and 2017-18 (p = 0.003) seasons. At d7 post-vaccination, higher magnitudes of cTfh activation were associated with increased clonality of cTfh TCR repertoire. The TCRs from vaccine-expanded clonotypes were identified and tracked longitudinally with several TCRs found to be present in both years. The transcriptomic profile of these expanded cTfh cells at the single cell level demonstrated overrepresentation of transcripts of genes involved in the type-I interferon pathway, pathways involved in gene expression, and antigen presentation and recognition. These results identify the expansion and transcriptomic profile of vaccine-induced cTfh cells important for B cell help.

Influenza Infection in Ferrets with SARS-CoV-2 Infection History.

Nonpharmaceutical interventions (NPIs) to contain the SARS-CoV-2 pandemic drastically reduced human-to-human interactions, decreasing the circulation of other respiratory viruses, as well. Consequently, influenza virus circulation, which is normally responsible for 3 to 5 million hospitalizations per year globally, was significantly reduced. With the downscaling of the NPI countermeasures, there is a concern for increased influenza disease, particularly in individuals suffering from postacute effects of SARS-CoV-2 infection. To investigate this, we performed a sequential influenza H1N1 infection 4 weeks after an initial SARS-CoV-2 infection in ferrets. Upon H1N1 infection, ferrets that were previously infected with SARS-CoV-2 showed an increased tendency to develop clinical signs, compared to the control H1N1-infected animals. A histopathological analysis indicated only a slight increase for type II pneumocyte hyperplasia and bronchitis. Thus, the effects of the sequential infection appeared minor. However, ferrets were infected with B.1.351-SARS-CoV-2, the beta variant of concern, which replicated poorly in our model. The histopathology of the respiratory organs was mostly resolved 4 weeks after the SARS-CoV-2 infection, with only reminiscent histopathological features in the upper respiratory tract. Nevertheless, SARS-CoV-2 specific cellular and humoral responses were observed, confirming an established infection. On account of a modest trend toward the enhancement of the influenza disease, even upon a mild SARS-CoV-2 infection, our findings suggest that a stronger SARS-CoV-2 infection and its consequent, long-term effects could have a greater impact on the outcome of disease after a sequential influenza infection. Hence, the influenza vaccination of individuals suffering from postacute SARS-CoV-2 infection effects may be considered an avertible measure for such a scenario. IMPORTANCE During the COVID-19 pandemic, the use of face masks, social distancing, and isolation were effective not only in decreasing the circulation of SARS-CoV-2 but also in reducing other respiratory viruses, such as influenza. With fewer restrictions currently in place, influenza is slowly returning. In the meantime, people who are still suffering from long-COVID could be more vulnerable to an influenza virus infection and could develop a more severe influenza disease. This study provides directions to the effect of a previous SARS-CoV-2 exposure on influenza disease severity in a ferret model. This model is highly valuable to test sequential infections under controlled settings for translation to humans. We could not induce clear long-term COVID-19 effects, as the SARS-CoV-2 infections in the ferrets were mild. However, we still observed a slight increase in influenza disease severity compared to ferrets that had not encountered SARS-CoV-2 before. Therefore, it may be advisable to include long-COVID patients as a risk group for influenza vaccination.

Tempel: time-series mutation prediction of influenza A viruses via attention-based recurrent neural networks.

Influenza viruses are persistently threatening public health, causing annual epidemics and sporadic pandemics. The evolution of influenza viruses remains to be the main obstacle in the effectiveness of antiviral treatments due to rapid mutations. The goal of this work is to predict whether mutations are likely to occur in the next flu season using historical glycoprotein hemagglutinin sequence data. One of the major challenges is to model the temporality and dimensionality of sequential influenza strains and to interpret the prediction results. In this article, we propose an efficient and robust time-series mutation prediction model (Tempel) for the mutation prediction of influenza A viruses. We first construct the sequential training samples with splittings and embeddings. By employing recurrent neural networks with attention mechanisms, Tempel is capable of considering the historical residue information. Attention mechanisms are being increasingly used to improve the performance of mutation prediction by selectively focusing on the parts of the residues. A framework is established based on Tempel that enables us to predict the mutations at any specific residue site. Experimental results on three influenza datasets show that Tempel can significantly enhance the predictive performance compared with widely used approaches and provide novel insights into the dynamics of viral mutation and evolution. The datasets, source code and supplementary documents are available at: https://drive.google.com/drive/folders/15WULR5__6k47iRotRPl3H7ghi3RpeNXH. Supplementary data are available at Bioinformatics online.

2750. Sequential Influenza A H1N1 and Influenza A H3N2 Challenge Infections in Healthy Volunteers

BackgroundSeasonal influenza causes significant annual morbidity and mortality. The effects of yearly exposures on immunity are not clear and recent observations have demonstrated that long lasting protection against a matched strain may not naturally occur. The 2018–2019 influenza season consisted of an initial peak of H1N1 infections followed by a wave of H3N2 infections. These consecutive waves raise questions about how influenza immunity is affected by sequential exposure to different influenza strains. Challenge studies provide a unique opportunity to study this phenomenon. Here we describe a subset of participants who were sequentially infected in two separate challenge studies with wild-type H1N1 and H3N2 viruses.MethodsHealthy volunteers completed two sequential influenza challenge studies at the NIH Clinical Center. Participants were inoculated with reverse genetics, cell-based, GMP wild-type influenza viruses, A(H1N1)pdm09 and A(H3N2) strains. Participants remained isolated in the hospital for a minimum of 9 days and were monitored daily for viral shedding and clinical symptoms. After discharge, participants were followed for 2 months.ResultsBetween 2014 and 2017, 14 healthy volunteers were exposed to Influenza A(H1N1) and Influenza A(H3N2). Time between infections ranged from 2 months to 2 years. Thirteen (93%) participants developed confirmed influenza infection after H1N1 challenge and 9 (64%) after H3N2 challenge. Eight (57%) participants developed confirmed infections after both exposures. Variable degrees of symptoms, shedding, and disease severity were observed. Systemic antibody responses to the HA and NA of both H1N1 and H3N2 varied over time during these sequential infections.ConclusionMore than half of all participants who completed 2 sequential H1N1 and H3N2 challenge studies demonstrated confirmed infection to both viruses. These sequential infections had varying effects on the disease experienced and the immunity that developed after infection. These observations are important in understanding the impact of sequential exposures on influenza immunity.Disclosures All authors: No reported disclosures.

Influenza A Reinfection in Sequential Human Challenge: Implications for Protective Immunity and "Universal" Vaccine Development.

Identification of correlates of protection against human influenza A virus infection is important in development of broadly protective ("universal") influenza vaccines. Certain assumptions underlie current vaccine developmental strategies, including that infection with a particular influenza A virus should offer long-term or lifelong protection against that strain, preventing reinfection. In this study we report observations made when 7 volunteers participated in sequential influenza challenge studies where they were challenged intranasally using the identical influenza A(H1N1)pdm09 virus approximately 1 year apart. We evaluate and describe the outcomes of these 7 rechallenge participants and discuss what these results may suggest about correlates of protection and development of more broadly protective influenza vaccines. Seven participants were enrolled in 2 viral challenge studies at 7.5- to 18.5-month intervals. Both challenge studies used the identical lot of influenza A (H1N1)pdm09 virus administered intranasally. We evaluated pre- and postchallenge hemagglutination inhibition, neuraminidase inhibition, and stalk antibody titers; peripheral blood leukocyte host gene expression response profiles; daily viral detection via nasal wash; and clinical signs and symptoms. At least 3 of 7 participants demonstrated confirmed laboratory evidence of sequential infection, with 5 of 7 demonstrating clinical evidence. The data presented in this report demonstrate that sequential infection with the identical influenza A virus can occur and suggest it may not be rare. These data raise questions about immune memory responses in an acute superficial respiratory mucosal infection and their implications in development of broadly protective influenza vaccines. Further investigation of these observations is warranted. NCT01646138; NCT01971255.

Effects of Sequential Influenza A(H1N1)pdm09 Vaccination on Antibody Waning.

Antibody waning following influenza vaccination has been repeatedly evaluated, but waning has rarely been studied in the context of longitudinal vaccination history. We developed a Bayesian hierarchical model to assess the effects of sequential influenza A(H1N1)pdm09 vaccination on hemagglutination inhibition antibody boosting and waning in a longitudinal cohort of older children and adults from 2011 to 2016, a period during which the A(H1N1)pdm09 vaccine strain did not change. Antibody measurements from 2057 serum specimens longitudinally collected from 388 individuals were included. Average postvaccination antibody titers were similar across successive vaccinations, but the rate of antibody waning increased with each vaccination. The antibody half-life was estimated to decrease from 32 months (95% credible interval [CrI], 22-61 months) following first vaccination to 9 months (95% CrI, 7-15 months) following a seventh vaccination. Although the rate of antibody waning increased with successive vaccination, the estimated antibody half-life was longer than a typical influenza season even among the most highly vaccinated. This supports current recommendations for vaccination at the earliest opportunity. Patterns of boosting and waning might be different with the influenza A(H3N2) subtype, which evolves more rapidly and has been most associated with reduced effectiveness following repeat vaccination.

Cross-Protective Immune Responses Induced by Sequential Influenza Virus Infection and by Sequential Vaccination With Inactivated Influenza Vaccines.

Sequential infection with antigenically distinct influenza viruses induces cross-protective immune responses against heterologous virus strains in animal models. Here we investigated whether sequential immunization with antigenically distinct influenza vaccines can also provide cross-protection. To this end, we compared immune responses and protective potential against challenge with A(H1N1)pdm09 in mice infected sequentially with seasonal A(H1N1) virus followed by A(H3N2) virus or immunized sequentially with whole inactivated virus (WIV) or subunit (SU) vaccine derived from these viruses. Sequential infection provided solid cross-protection against A(H1N1)pdm09 infection while sequential vaccination with WIV, though not capable of preventing weight loss upon infection completely, protected the mice from reaching the humane endpoint. In contrast, sequential SU vaccination did not prevent rapid and extensive weight loss. Protection correlated with levels of cross-reactive but non-neutralizing antibodies of the IgG2a subclass, general increase of memory T cells and induction of influenza-specific CD4+ and CD8+ T cells. Adoptive serum transfer experiments revealed that despite lacking neutralizing activity, serum antibodies induced by sequential infection protected mice from weight loss and vigorous virus growth in the lungs upon A(H1N1)pdm09 virus challenge. Antibodies induced by WIV vaccination alleviated symptoms but could not control virus growth in the lung. Depletion of T cells prior to challenge revealed that CD8+ T cells, but not CD4+ T cells, contributed to cross-protection. These results imply that sequential immunization with WIV but not SU derived from antigenically distinct viruses could alleviate the severity of infection caused by a pandemic and may improve protection to unpredictable seasonal infection.

The Doctrine of Original Antigenic Sin: Separating Good From Evil

The term “original antigenic sin” was coined approximately 60 years ago to describe the imprinting by the initial first influenza A virus infection on the antibody response to subsequent vaccination. These studies did not suggest a reduction in the response to current antigens but instead suggested anamnestic recall of antibody to earlier influenza virus strains. Then, approximately 40 years ago, it was observed that sequential influenza vaccination might lead to reduced vaccine effectiveness (VE). This conclusion was largely dismissed after an experimental study involving sequential administration of then-standard influenza vaccines. Recent observations have provided convincing evidence that reduced VE after sequential influenza vaccination is a real phenomenon. We propose that such reduction in VE be termed “negative antigenic interaction,” given that there is no age cohort effect. In contrast, the potentially positive protective effect of early influenza virus infection later in life continues to be observed. It is essential that we understand better the immunologic factors underlying both original antigenic sin and negative antigenic interaction, to support development of improved influenza vaccines and vaccination strategies.

Progesterone-Based Contraceptives Reduce Adaptive Immune Responses and Protection against Sequential Influenza A Virus Infections.

In addition to their intended use, progesterone (P4)-based contraceptives promote anti-inflammatory immune responses, yet their effects on the outcome of infectious diseases, including influenza A virus (IAV) infection, are rarely evaluated. To evaluate their impact on immune responses to sequential IAV infections, adult female mice were treated with placebo or one of two progestins, P4 or levonorgestrel (LNG), and infected with a mouse-adapted H1N1 (maH1N1) virus. Treatment with P4 or LNG reduced morbidity but had no effect on pulmonary virus titers during primary H1N1 infection compared to placebo treatment. In serum and bronchoalveolar lavage fluid, total anti-IAV IgG and IgA titers and virus-neutralizing antibody titers but not hemagglutinin stalk antibody titers were lower in progestin-treated mice than placebo-treated mice. Females were challenged 6 weeks later with either an maH1N1 drift variant (maH1N1dv) or maH3N2 IAV. The level of protection following infection with the maH1N1dv was similar among all groups. In contrast, following challenge with maH3N2, progestin treatment reduced survival as well as the numbers and activity of H1N1- and H3N2-specific memory CD8+ T cells, including tissue-resident cells, compared with placebo treatment. In contrast to primary IAV infection, progestin treatment increased the titers of neutralizing and IgG antibodies against both challenge viruses compared with those achieved with placebo treatment. While the immunomodulatory properties of progestins protected immunologically naive female mice from the severe outcomes from IAV infection, it made them more susceptible to secondary challenge with a heterologous IAV, despite improving their antibody responses against a secondary IAV infection. Taken together, the immunomodulatory effects of progestins differentially regulate the outcome of infection depending on exposure history.IMPORTANCE The impact of hormone-based contraceptives on the outcome of infectious diseases outside the reproductive tract is rarely considered. Using a mouse model, we have made the novel observation that treatment with either progesterone or a synthetic analog found in hormonal contraceptives, levonorgestrel, impacts sequential influenza A virus infection by modulating antibody responses and decreasing the numbers and activity of memory CD8+ T cells. Progestins reduced the antibody responses during primary H1N1 virus infection but increased antibody titers following a sequential infection with either an H1N1 drift variant or an H3N2 virus. Following challenge with an H3N2 virus, female mice treated with progestins experienced greater mortality with increased pulmonary inflammation and reduced numbers and activity of CD8+ T cells. This study suggests that progestins significantly affect adaptive immune responses to influenza A virus infection, with their effect on the outcome of infection depending on exposure history.

Sequential Infection in Ferrets with Antigenically Distinct Seasonal H1N1 Influenza Viruses Boosts Hemagglutinin Stalk-Specific Antibodies.

Broadly reactive antibodies targeting the conserved hemagglutinin (HA) stalk region are elicited following sequential infection or vaccination with influenza viruses belonging to divergent subtypes and/or expressing antigenically distinct HA globular head domains. Here, we demonstrate, through the use of novel chimeric HA proteins and competitive binding assays, that sequential infection of ferrets with antigenically distinct seasonal H1N1 (sH1N1) influenza virus isolates induced an HA stalk-specific antibody response. Additionally, stalk-specific antibody titers were boosted following sequential infection with antigenically distinct sH1N1 isolates in spite of preexisting, cross-reactive, HA-specific antibody titers. Despite a decline in stalk-specific serum antibody titers, sequential sH1N1 influenza virus-infected ferrets were protected from challenge with a novel H1N1 influenza virus (A/California/07/2009), and these ferrets poorly transmitted the virus to naive contacts. Collectively, these findings indicate that HA stalk-specific antibodies are commonly elicited in ferrets following sequential infection with antigenically distinct sH1N1 influenza virus isolates lacking HA receptor-binding site cross-reactivity and can protect ferrets against a pathogenic novel H1N1 virus. The influenza virus hemagglutinin (HA) is a major target of the humoral immune response following infection and/or seasonal vaccination. While antibodies targeting the receptor-binding pocket of HA possess strong neutralization capacities, these antibodies are largely strain specific and do not confer protection against antigenic drift variant or novel HA subtype-expressing viruses. In contrast, antibodies targeting the conserved stalk region of HA exhibit broader reactivity among viruses within and among influenza virus subtypes. Here, we show that sequential infection of ferrets with antigenically distinct seasonal H1N1 influenza viruses boosts the antibody responses directed at the HA stalk region. Moreover, ferrets possessing HA stalk-specific antibody were protected against novel H1N1 virus infection and did not transmit the virus to naive contacts.

Influenza viral load and peramivir kinetics after single administration and proposal of regimens for peramivir administration against resistant variants.

We estimated the efficacy of the current single administration of peramivir on the basis of peramivir pharmacokinetics in the upper respiratory tract (URT) and determined the predictive peramivir concentration-time curve to assess its efficacy against viruses with decreased susceptibility to neuraminidase inhibitors. Serum, nasal swab, or aspiration samples were collected from 28 patients treated with 10 mg/kg body weight peramivir. The sequential influenza viral RNA load and susceptibility after peramivir administration were measured using a quantitative real-time reverse transcription-PCR and neuraminidase inhibition assay. The peramivir concentrations in the serum and URT after a single administration at 10 mg/kg were measured, and the predictive blood and URT peramivir concentration-time curves were determined to assess various administration regimens against resistant variants. The peramivir concentration decreased to <0.1% of the maximum concentration of drug in serum (Cmax) at 24 h after administration. Rapid elimination of peramivir from the URT by 48 h after administration may contribute to an increase in the influenza A viral load after day 3 but not to a decrease in the influenza B viral load, despite the absence of a decrease in the susceptibility to peramivir. A longer maintenance of a high level of peramivir in the URT is expected by divided administration rather than once-daily administration. When no clinical improvement is observed in patients with normal susceptibility influenza A and B, peramivir readministration should be considered. In severe cases caused by resistant variants, better inhibitory effectiveness and less frequent adverse events are expected by divided administration rather than once-daily administration with an increased dosage.

Impaired heterologous immunity in aged ferrets during sequential influenza A H1N1 infection

The major burden of influenza morbidity resides within the elderly population. The challenge managing influenza-associated illness in the elderly is the decline of immune function, where mechanisms leading to immunological senescence have not been elucidated. To better represent the immune environment, we investigated clinical morbidity and immune function during sequential homologous and heterologous H1N1 influenza infection in an aged ferret model. Our findings demonstrated experimentally that aged ferrets had significant morbidity during monosubtypic heterologous 2° challenge with significant weight loss and respiratory symptoms. Furthermore, increased clinical morbidity was associated with slower and shorter hemagglutinin antibody generation and attenuated type 1 T-cell gene responses in peripheral blood. These results revealed dampened immune activation during sequential influenza infection in aged ferrets. With the presence of an aged model, dissecting clinical morbidity, viral dynamics and immune response during influenza infection will aid the development of future prophylactics such as age specific influenza vaccines.

Sequential Influenza B Viral Load and Susceptibility in Children Treated With Oseltamivir and Zanamivir

The objective of this study was to estimate the efficacy of the neuraminidase (NA) inhibitors (NAIs) oseltamivir and zanamivir for decreasing viral load and to investigate whether NAI treatment decreases viral susceptibility to NAIs over time in children with influenza B virus infection. Of 27 patients with influenza B virus infection, 8 and 9 were treated with oseltamivir and zanamivir, respectively, whereas 10 received no NAI. Nasal aspiration samples, collected every morning until negative antigen results in 2 consecutive samples were observed, were subjected to viral load measurements by quantitative real-time reverse transcription polymerase chain reaction and viral susceptibility to NAI by NA inhibition assays. Viral load decreased in both the oseltamivir and zanamivir groups by day 2 but increased in the no-NAI treatment group. Viral load in the oseltamivir and zanamivir groups on day 5 was 2.6% and 9.2% of that on day 0, respectively, whereas it was 26.4% in the no-NAI treatment group. Mean 50% inhibitory concentration (IC50) values of oseltamivir and zanamivir in the no-NAI treatment group were 5.0-6.6 and 1.3-1.8 nM, respectively. Mean IC50 values of oseltamivir and zanamivir in patients treated with oseltamivir and zanamivir were 3.9-8.8 and 1.3-1.8 nM, respectively. No major decrease in viral susceptibility to NAIs was observed during or after NAI treatment. NAI treatment was effective for inhibiting viral replication during the early days of illness and did not decrease viral susceptibility to NAIs in patients with influenza B virus infection.

Cutting Edge: Heterosubtypic Influenza Infection Antagonizes Elicitation of Immunological Reactivity to Hemagglutinin

Influenza-specific immunity in humans is unique because there are repeated exposures to viral strains containing genetically conserved epitopes recruiting memory CD4 T cells and novel epitopes stimulating naive CD4 T cells, possibly resulting in competition between memory and naive lymphocytes. In this study, we evaluated the effect of this competition on CD4 T cell and B cell response specificity using a murine model of sequential influenza infection. We found striking and selective decreases in CD4 T cell reactivity to nonconserved hemagglutinin (HA) epitopes following secondary influenza infection. Surprisingly, this shift in CD4 T cell specificity was associated with dramatic decreases in HA-specific Ab. These results suggest that repeated exposure to influenza viruses and vaccines containing conserved internal proteins may have unintended and negative consequences on the ability to induce HA-specific Ab to novel pandemic strains of influenza. These finding could have important implications on pandemic influenza preparedness strategies.

Sequential Seasonal H1N1 Influenza Virus Infections Protect Ferrets against Novel 2009 H1N1 Influenza Virus

Individuals <60 years of age had the lowest incidence of infection, with ~25% of these people having preexisting, cross-reactive antibodies to novel 2009 H1N1 influenza. Many people >60 years old also had preexisting antibodies to novel H1N1. These observations are puzzling because the seasonal H1N1 viruses circulating during the last 60 years were not antigenically similar to novel H1N1. We therefore hypothesized that a sequence of exposures to antigenically different seasonal H1N1 viruses can elicit an antibody response that protects against novel 2009 H1N1. Ferrets were preinfected with seasonal H1N1 viruses and assessed for cross-reactive antibodies to novel H1N1. Serum from infected ferrets was assayed for cross-reactivity to both seasonal and novel 2009 H1N1 strains. These results were compared to those of ferrets that were sequentially infected with H1N1 viruses isolated prior to 1957 or more-recently isolated viruses. Following seroconversion, ferrets were challenged with novel H1N1 influenza virus and assessed for viral titers in the nasal wash, morbidity, and mortality. There was no hemagglutination inhibition (HAI) cross-reactivity in ferrets infected with any single seasonal H1N1 influenza viruses, with limited protection to challenge. However, sequential H1N1 influenza infections reduced the incidence of disease and elicited cross-reactive antibodies to novel H1N1 isolates. The amount and duration of virus shedding and the frequency of transmission following novel H1N1 challenge were reduced. Exposure to multiple seasonal H1N1 influenza viruses, and not to any single H1N1 influenza virus, elicits a breadth of antibodies that neutralize novel H1N1 even though the host was never exposed to the novel H1N1 influenza viruses.

Hemagglutinin Stalk-Reactive Antibodies Are Boosted following Sequential Infection with Seasonal and Pandemic H1N1 Influenza Virus in Mice

Previously, it has been shown that infection in humans with the pandemic swine influenza virus induces antibodies with specificity to the stalk domain of the viral hemagglutinin. Following the generation of these data, we sought to recapitulate these findings in the mouse model by sequential influenza virus infection. Mice that were inoculated with a seasonal influenza H1N1 virus followed by infection with a pandemic H1N1 strain produced higher antihemagglutinin stalk antibody titers than mice sequentially infected with drifted seasonal strains. In order to achieve antibody titers of comparable magnitude using sequential infection, mice had to be infected with 100- to 1,000-fold more of the drifted seasonal virus. The antistalk antibodies produced by these infections were influenza virus neutralizing, which illustrates the utility of the mouse model in which to study this interaction between virus and host.

Influenza and bacterial pneumonia - constant companions

Sequential or concomitant influenza and bacterial pneumonia are two common syndromes seen in community-acquired pneumonia. Inadequacies of diagnostic testing make separating simple pneumonia with either bacteria or influenza from concomitant or sequential influenza with both microorganisms difficult, although the novel 2009 H1N1 epidemic may improve the availability of molecular testing for viruses. Given the frequency of viral pneumonia and diagnostic limitations, empirical antivirals may be underutilized in community-acquired pneumonia. Thankfully, increasingly effective vaccines appear to disrupt this synergistic relationship.

A model of meningococcal bacteremia after respiratory superinfection in influenza A virus-infected mice

We developed a model of sequential influenza A virus (IAV)– Neisseria meningitidis serogroup C (Nm) infection in BALB/c mice. Mice infected intranasally with a sublethal IAV dose (260 pfu) were superinfected intranasally with Nm. Fatal meningococcal pneumonia and bacteremia were observed in IAV-infected mice superinfected with Nm on day 7, but not in those superinfected on day 10. The susceptibility of mice to Nm superinfection was correlated with the peak interferon-γ production in the lungs and decrease in IAV load. After Nm challenge, both IAV-infected and uninfected control mice produced the inflammatory cytokines interleukin (IL)-1 and IL-6. However, IL-10 was detected in susceptible mice superinfected on day 7 after IAV infection, but not in resistant mice. This model of dual IAV–Nm infection was also used to evaluate the role of bacterial virulence factors in the synthesis of the capsule. A capsule-defective mutant was cleared from the lungs, whereas a mutant inactivated for the crgA gene, negatively regulating expression of the pili and capsule, upon contact with host cells, retained invasiveness. Therefore, this model of meningococcal disease in adult mice reproduces the pathogenesis of human meningococcemia with fatal sepsis, and is useful for analyzing known or new genes identified in genomic studies.

Efficacy of zanamivir for chemoprophylaxis of nursing home influenza outbreaks

Despite vaccination, influenza remains a common cause of morbidity in nursing homes. Chemoprophylaxis of residents with currently available antivirals is not always effective and new agents effective against both influenza A and B are needed. In a randomized, unblinded pilot study, we compared 14 day chemoprophylaxis with zanamivir, an antiviral which inhibits influenza neuraminidase, to standard of care during sequential influenza A and influenza B outbreaks in a 735 bed nursing home. Influenza A outbreaks were declared on 6 14 epidemic units. Sixty-five volunteers on four epidemic units were randomized to zanamivir and on two epidemic units, 23 volunteers were randomized to rimantadine. During the 14 days of prophylaxis, only four new febrile respiratory illnesses were detected. One volunteer receiving rimantadine prophylaxis developed laboratory-confirmed influenza. Influenza B outbreaks were declared on 3 14 epidemic units. Thirty-five volunteers on two epidemic units were randomized to zanamivir and 18 volunteers on one epidemic unit were randomized to no drug. During the 14 days of prophylaxis, only one new febrile respiratory illness was detected. One volunteer randomized to receive no drug developed laboratory-confirmed influenza. Zanamivir appears comparably effective to standard of care in preventing influenza-like illness and laboratory-confirmed influenza in nursing homes, but requires further testing.

Analysis of influenza A H3N2 strains isolated in England during 1995–1996 using polymerase chain reaction restriction

A polymerase chain reaction-restriction (PCR-restriction) endonuclease assay was developed to allow rapid analysis of influenza A H3N2 viruses circulating in England during 1995-1996. Restriction endonuclease digestion with two enzymes of amplicons derived from PCR of the HA1 portion of the influenza haemagglutinin (HA) gene was able to differentiate antigenically similar influenza strains into two groups. Group I variants were similar genetically to the 1995/96 vaccine strain, A/Johannesburg/33/94, whereas the HA sequences of Group II variants were similar genetically to the reference virus A/Thessaloniki/1/95. Of the 700 England A H3N2 strains isolated between February 1995 and the end of April 1996, 384 were analysed by this method. PCR-restriction analysis of sequential influenza isolates revealed a temporal alteration in prevalence of two variants. Groups I and II variants cocirculated with equal frequency during a period of sporadic influenza activity, but following the onset of epidemic influenza activity in 1995, only Group II variants were detected. PCR- restriction analysis was found to be a rapid method for studying genetic variation which could be applied to a large number of samples and provide information about the direction of genetic drift in the HA gene of influenza virus.