Amy R. Iverson

Influenza enhances susceptibility to natural acquisition of and disease due to Streptococcus pneumoniae in ferrets.

The role of respiratory viruses in the transmission of Streptococcus pneumoniae is poorly understood. Key questions, such as which serotypes are most fit for transmission and disease and whether influenza virus alters these parameters in a serotype-specific manner, have not been adequately studied. In a novel model of transmission in ferrets, we demonstrated that pneumococcal transmission and disease were enhanced if donors had previously been infected with influenza virus. Bacterial titers in nasal wash, the incidence of mucosal and invasive disease, and the percentage of contacts that were infected all increased. In contact ferrets, viral infection increased their susceptibility to S. pneumoniae acquisition both in terms of the percentage infected and the distance over which they could acquire infection. These influenza-mediated effects on colonization, transmission, and disease were dependent on the pneumococcal strain. Overall, these data argue that the relationship between respiratory viral infections, acquisition of pneumococci, and development of disease in humans needs further study to be better understood.

Multiple Genotypes of Influenza B Virus Circulated between 1979 and 2003

ABSTRACT The segmented genome of influenza B virus allows exchange of gene segments between cocirculating strains. Through this process of reassortment, diversity is generated by the mixing of genes between viruses that differ in one or more gene segments. Phylogenetic and evolutionary analyses of all 11 genes of 31 influenza B viruses isolated from 1979 to 2003 were used to study the evolution of whole genomes. All 11 genes diverged into two new lineages prior to 1987. All genes except the NS1 gene were undergoing linear evolution, although the rate of evolution and the degree to which nucleotide changes translated into amino acid changes varied between lineages and by gene. Frequent reassortment generated 14 different genotypes distinct from the gene constellation of viruses circulating prior to 1979. Multiple genotypes cocirculated in some locations, and a sequence of reassortment events over time could not be established. The surprising diversity of the viruses, unrestricted mixing of lineages, and lack of evidence for coevolution of gene segments do not support the hypothesis that the reassortment process is driven by selection for functional differences.

Influenza Virus Primes Mice for Pneumonia From Staphylococcus aureus

Superinfections from Staphylococcus aureus following influenza are an increasing concern. We assessed several laboratory and clinical strains in a mouse coinfection model with influenza virus. A methicillin-resistant USA300 clone and several recent clinical strains from patients with necrotizing pneumonia caused high mortality following influenza virus infection in mice. Both viral and bacterial lung titers were enhanced during coinfections compared with single infections. However, differences in titers did not correspond with differences in disease outcomes in a comparison of superinfections from a highly pathogenic strain with those from a poorly pathogenic strain. These strains did differ, however, in expression of Panton-Valentine leukocidin and in the degree of inflammatory lung damage each engendered. The viral cytotoxin PB1-F2 contributed to the negative outcomes. These data suggest that additional study of specific bacterial virulence factors involved in the pathogenesis of inflammation and lung damage during coinfections is needed.

Novel Strategy to Prevent Otitis Media Caused by Colonizing Streptococcus pneumoniae

In early childhood, 70%–83% of children experience at least one episode of acute otitis media (AOM) [1,2]. Streptococcus pneumoniae is the most common bacterial agent identified as the causative agent of these infections, although there is increasing evidence that a variety of respiratory viruses play a prominent role in the development and pathogenesis of AOM [1]. Even though the heptavalent pneumococcal conjugate vaccine appears to be making an impact on the incidence of this disease among children in the United States, AOM (with more than 24 million diagnoses annually) remains the leading reason for physician visits and antibiotic prescriptions among preschool-aged children [2,3]. Frequent use of antibiotics for AOM has led to a vicious cycle of diminishing returns: increased exposure has led to increasing drug resistance, which in turn makes the infections more difficult to treat, necessitating new drugs and more treatment. Recently, purified bacteriophage (phage) cell wall hydrolases, or lysins, have shown promise as novel anti-infectives due to their ability to eradicate nasal carriage of gram-positive pathogens, particularly S. pneumoniae [4,5]. These highly active enzymes are produced by phages to disrupt the bacterial cell wall for the release of progeny phage. Here, we show that the Cpl-1 lysin, which is specific for S. pneumoniae [6], prevents AOM in a novel mouse model that mimics the natural pathogenesis of this common infection. Current animal models for AOM have critical limitations. Modeling AOM in mice requires invasive and artificial procedures to establish infection, and sacrifice of the animals to determine outcomes. Larger animals such as chinchillas and ferrets may develop infection by more natural routes, but use of these models is limited by their size and complexity [7,8]. Ideally, we wished to develop a non-invasive mouse model that was permissive of natural infection. We engineered a piliated strain of S. pneumoniae, known to efficiently colonize mucosal surfaces (a type 19F strain obtained from B. Henriques-Normark, ST16219F) [9], to express luciferase [10]. Groups of five mice maintained in a BL2 facility were infected intranasally with 1 × 105 or 1 × 106 colony-forming units (CFU) of this bioluminescent strain under light anesthesia with 2.5% inhaled isoflurane using an established infection model approved by the St. Jude Childrens Research Hospital animal care and use committee [11]. Animals were followed daily for development of infection for two weeks and thrice weekly for another four weeks. Within 72 hours of pneumococcal infection, 100% of mice (10/10) were visibly colonized with bacteria in the anterior portion of their nose, and 70% (7/10) had developed AOM. These infections of the middle ear all resolved by bioluminescent imaging within 48 hours, and no mice had evidence of AOM six days after challenge or later. Nasal colonization persisted for a median of 27 days (range 17–34 days). Around half of all children are colonized with S. pneumoniae [12]. Alteration of eustachian tube function or disruption of mucosal surfaces through viral infection allows colonizing bacteria to ascend into the middle ear, triggering AOM [1]. To model this phenomenon, we infected mice that had been stably colonized by pneumococcus with influenza virus and followed them for development of AOM. Although all mice had been colonized prior to infection with virus, 63% of virus-infected mice (19/30) developed AOM compared to 0% (0/10) of mice mock-infected with phosphate buffered saline (PBS) (Figure 1). Twenty-one of thirty mice in the virus group had experienced AOM after introduction of the bacteria in the first 72 hours post colonization (with resolution before viral challenge), while eight of ten mice in the PBS control group had experienced AOM with resolution (unpublished data). Both de novo and recurrent infections were seen in the virus-infected mice, with no correlation to whether they had previously had AOM. This is the first mouse model of AOM in which infection develops in a manner analogous to that observed in children. Figure 1 Visualization of Bioluminescent Bacteria Inside Live, Anesthetized Animals Shows the Induction of Otitis Media Using this novel and powerful model, we sought to test our hypothesis that reduction or elimination of colonizing pneumococci with purified Cpl-1 lysin [6] would prevent the development of AOM. Prior to infection with influenza virus, mice colonized with pneumococcus for seven days were treated twice four hours apart with either 1,000 ug of Cpl-1 intranasally or enzyme buffer (mock treatment). At the time of the second treatment, nine of ten animals (90%) had cleared the pneumococcus from the nose, compared to zero of ten (0%) treated with enzyme buffer. The Cpl-1 lysin was 100% effective in preventing AOM, as no animal treated with lysin developed a secondary bacterial infection following influenza infection, while eight of ten (80%) mice that were mock-treated developed AOM (Figure 2). In the one mouse in which colonization persisted despite lysin treatment, the amount of bacteria present decreased dramatically (78% reduction in flux of light through the nose). In addition, no mice treated with Cpl-1 and observed for the duration of these experiments developed toxicity or illness attributable to the enzyme as determined by clinical observation, weight loss, and histopathology (unpublished data). Figure 2 Treatment with Lysin Eliminates Colonization and Prevents the Development of Otitis Media This novel model of naturally developing AOM will be useful in studies of prevention and treatment of this important and common infection. Our data on the use of lysin in the model suggest that a strategy of decolonizing children of S. pneumoniae may prevent many cases of AOM, particularly those with chronic AOM. Importantly, these data also suggest that elimination or even reduction of resident pathogenic bacteria can prevent secondary bacterial complications of influenza. In support of this view, a recent study of a pediatric pneumococcal vaccine in South Africa showed a 31% decrease in the incidence of virus-associated pneumonia compared to controls [13]. Secondary bacterial infections account for much of the morbidity and approximately 25% of all deaths during seasonal epidemics of influenza, as well as 50%–95% of deaths during pandemics of influenza [14]. It is very likely that a reduction in pneumococcal colonization in susceptible populations such as infants and the elderly during a pandemic or annual influenza outbreak could result in a concomitant reduction in morbidity and mortality. With worldwide concerns over a potentially incipient pandemic with highly pathogenic influenza viruses of the H5N1 subtype, further study of novel therapeutics such as phage-derived lysins to prevent these infections is warranted.

Contribution of Vaccine-Induced Immunity toward either the HA or the NA Component of Influenza Viruses Limits Secondary Bacterial Complications

ABSTRACT Secondary bacterial infections contribute to morbidity and mortality from influenza. Vaccine effectiveness is typically assessed using prevention of influenza, not secondary infections, as an endpoint. We vaccinated mice with formalin-inactivated influenza virus vaccine preparations containing disparate HA and NA proteins and demonstrated an ability to induce the appropriate anti-HA and anti-NA immune profiles. Protection from both primary viral and secondary bacterial infection was demonstrated with vaccine-induced immunity directed toward either the HA or the NA. This finding suggests that immunity toward the NA component of the virion is desirable and should be considered in generation of influenza vaccines.

Safety and Immunogenicity of Live Attenuated and Inactivated Influenza Vaccines in Children With Cancer

BACKGROUND The safety and immunogenicity of live, attenuated influenza vaccine (LAIV) has not been compared to that of the standard trivalent inactivated vaccine (TIV) in children with cancer. METHODS Randomized study of LAIV versus TIV in children with cancer, age 2-21 years, vaccinated according to recommendations based on age and prior vaccination. Data on reactogenicity and other adverse events and blood and nasal swab samples were obtained following vaccination. RESULTS Fifty-five eligible subjects (mean age, 10.4 years) received vaccine (28 LAIV/27 TIV). Both vaccines were well tolerated. Rhinorrhea reported within 10 days of vaccination was similar in both groups (36% LAIV vs 33% TIV, P > .999). Ten LAIV recipients shed virus; the latest viral shedding was detected 7 days after vaccination. Immunogenicity data were available for 52 subjects, or 26 in each group. TIV induced significantly higher postvaccination geometric mean titers against influenza A viruses (P < .001), greater seroprotection against influenza A/H1N1 (P = .01), and greater seroconversion against A/H3N2 (P = .004), compared with LAIV. No differences after vaccination were observed against influenza B viruses. CONCLUSIONS As expected, serum antibody response against influenza A strains were greater with TIV than with LAIV in children with cancer. Both vaccines were well tolerated, and prolonged viral shedding after LAIV was not detected. CLINICAL TRIALS REGISTRATION NCT00906750.

Naturally occurring swine influenza A virus PB1-F2 phenotypes that contribute to superinfection with Gram-positive respiratory pathogens.

ABSTRACT A combination of viral, bacterial, and host factors contributes to the severity and overall mortality associated with influenza virus-bacterium superinfections. To date, the virulence associated with the recently identified influenza virus protein PB1-F2 has been largely defined using models of primary influenza virus infection, with only limited assessment in models of Streptococcus pneumoniae superinfection. Specifically, these studies have incorporated isogenic viruses that differ in the PB1-F2 expressed, but there is still knowledge to be gained from evaluation of natural variants derived from a nonhuman host species (swine). Using this rationale, we developed the hypothesis that naturally occurring viruses expressing variants of genes, like the PB1-F2 gene, can be associated with the severity of secondary bacterial infections. To test this hypothesis, we selected viruses expressing variants in PB1-F2 and evaluated outcomes from superinfection with three distinct Gram-positive respiratory pathogens: Streptococcus pneumoniae, Staphylococcus aureus, and Streptococcus pyogenes. Our results demonstrate that the amino acid residues 62L, 66S, 75R, 79R, and 82L, previously proposed as molecular signatures of PB1-F2 virulence for influenza viruses in the setting of bacterial superinfection, are broadly associated with enhanced pathogenicity in swine in a bacterium-specific manner. Furthermore, truncated PB1-F2 proteins can preferentially increase mortality when associated with Streptococcus pyogenes superinfection. These findings support efforts to increase influenza virus surveillance to consider viral genotypes that could be used to predict increased severity of superinfections with specific Gram-positive respiratory pathogens.

A live-attenuated pneumococcal vaccine elicits CD4+ T-cell dependent class switching and provides serotype independent protection against acute otitis media

Acute otitis media (AOM) caused by Streptococcus pneumoniae remains one of the most common infectious diseases worldwide despite widespread vaccination. A major limitation of the currently licensed pneumococcal vaccines is the lack of efficacy against mucosal disease manifestations such as AOM, acute bacterial sinusitis and pneumonia. We sought to generate a novel class of live vaccines that (1) retain all major antigenic virulence proteins yet are fully attenuated and (2) protect against otitis media. A live vaccine candidate based on deletion of the signal recognition pathway component ftsY induced potent, serotype‐independent protection against otitis media, sinusitis, pneumonia and invasive pneumococcal disease. Protection was maintained in animals coinfected with influenza virus, but was lost if mice were depleted of CD4+ T cells at the time of vaccination. The live vaccine induced a strong serum IgG2a and IgG2b response that correlated with CD4+ T‐cell mediated class switching. Deletion of genes required for microbial adaptation to the host environment is a novel live attenuated vaccine strategy yielding the first experimental vaccine effective against pneumococcal otitis media.

The platelet activating factor receptor is not required for exacerbation of bacterial pneumonia following influenza.

Pneumonia caused by Streptococcus pneumoniae is a significant cause of morbidity and mortality during influenza virus epidemics. We had previously advanced the hypothesis that interactions of pneumococcus with the receptor for platelet activating factor (PAFR) in the lung were facilitated by antecedent influenza virus infection and play a major role in the pathogenesis of bacterial superinfections. Although influenza enhanced the adherence of pneumococci to respiratory epithelial cells in vitro, chemical or antibody-mediated blockade of the PAFR did not affect adherence. In agreement with these data, mice lacking PAFR had similar bacterial loads within the lung compartment when compared to heterozygous littermates and were not protected from secondary pneumococcal pneumonia after influenza. Lack of support for this hypothesis and the observation of enhanced inflammation during secondary pneumococcal pneumonia in mice lacking PAFR may moderate enthusiasm for treatment strategies targeting the interaction of bacteria with PAFR.

RelA Mutant Enterococcus faecium with Multiantibiotic Tolerance Arising in an Immunocompromised Host

ABSTRACT Serious bacterial infections in immunocompromised patients require highly effective antibacterial therapy for cure, and thus, this setting may reveal novel mechanisms by which bacteria circumvent antibiotics in the absence of immune pressure. Here, an infant with leukemia developed vancomycin-resistant Enterococcus faecium (VRE) bacteremia that persisted for 26 days despite appropriate antibiotic therapy. Sequencing of 22 consecutive VRE isolates identified the emergence of a single missense mutation (L152F) in relA, which constitutively activated the stringent response, resulting in elevated baseline levels of the alarmone guanosine tetraphosphate (ppGpp). Although the mutant remained susceptible to both linezolid and daptomycin in clinical MIC testing and during planktonic growth, it demonstrated tolerance to high doses of both antibiotics when growing in a biofilm. This biofilm-specific gain in resistance was reflected in the broad shift in transcript levels caused by the mutation. Only an experimental biofilm-targeting ClpP-activating antibiotic was able to kill the mutant strain in an established biofilm. The relA mutation was associated with a fitness trade-off, forming smaller and less-well-populated biofilms on biological surfaces. We conclude that clinically relevant relA mutations can emerge during prolonged VRE infection, causing baseline activation of the stringent response, subsequent antibiotic tolerance, and delayed eradication in an immunocompromised state. IMPORTANCE The increasing prevalence of antibiotic-resistant bacterial pathogens is a major challenge currently facing the medical community. Such pathogens are of particular importance in immunocompromised patients as these individuals may favor emergence of novel resistance determinants due to lack of innate immune defenses and intensive antibiotic exposure. During the course of chemotherapy, a patient developed prolonged bacteremia with vancomycin-resistant Enterococcus faecium that failed to clear despite multiple front-line antibiotics. The consecutive bloodstream isolates were sequenced, and a single missense mutation identified in the relA gene, the mediator of the stringent response. Strains harboring the mutation had elevated baseline levels of the alarmone and displayed heightened resistance to the bactericidal activity of multiple antibiotics, particularly in a biofilm. Using a new class of compounds that modulate ClpP activity, the biofilms were successfully eradicated. These data represent the first clinical emergence of mutations in the stringent response in vancomycin-resistant entereococci. The increasing prevalence of antibiotic-resistant bacterial pathogens is a major challenge currently facing the medical community. Such pathogens are of particular importance in immunocompromised patients as these individuals may favor emergence of novel resistance determinants due to lack of innate immune defenses and intensive antibiotic exposure. During the course of chemotherapy, a patient developed prolonged bacteremia with vancomycin-resistant Enterococcus faecium that failed to clear despite multiple front-line antibiotics. The consecutive bloodstream isolates were sequenced, and a single missense mutation identified in the relA gene, the mediator of the stringent response. Strains harboring the mutation had elevated baseline levels of the alarmone and displayed heightened resistance to the bactericidal activity of multiple antibiotics, particularly in a biofilm. Using a new class of compounds that modulate ClpP activity, the biofilms were successfully eradicated. These data represent the first clinical emergence of mutations in the stringent response in vancomycin-resistant entereococci.