Mariana Baz

Characterization of Multidrug-Resistant Influenza A/H3N2 Viruses Shed during 1 Year by an Immunocompromised Child

BACKGROUND Development of influenza drug resistance is an important problem in immunocompromised children that could result in treatment failure and viral transmission to others. METHODS A total of 17 influenza A/H3N2 isolates were recovered over a period of 1 year from an immunocompromised child who was initially treated with oseltamivir and then with amantadine and zanamivir for viral pneumonitis. Drug susceptibility phenotypes to oseltamivir, zanamivir, and peramivir were evaluated by neuraminidase (NA) inhibition assays, and sequence analysis of key viral genes (i.e., M2, NA, and hemagglutinin [HA]) was performed. The impact of NA mutations identified in oseltamivir-resistant isolates was analyzed using recombinant NA proteins. RESULTS An influenza A variant with NA mutations E59G, E119V, and I222V was first detected after 38 days of oseltamivir treatment. In an NA inhibition assay, this variant was 274 times more resistant to oseltamivir than the original isolate but was susceptible to zanamivir. The I222V substitution enhanced the level of oseltamivir resistance that was primarily conferred by the E119V mutation in recombinant NA proteins. Remarkably, the E119V mutation persisted for 8 months after cessation of oseltamivir. Amantadine therapy led to rapid emergence of the M2 mutation S31N, which is known to confer amantadine resistance. The patient shed the virus intermittently while receiving nebulized zanamivir therapy despite the absence of a resistance phenotype, which could be the result of nonoptimal drug delivery and impaired host immunity. CONCLUSIONS This study highlights the potential for emergence and persistence of multidrug-resistant influenza isolates in immunocompromised subjects even after cessation of treatment, reinforcing the need for development of new anti-influenza compounds.

Development of a universal influenza A vaccine based on the M2e peptide fused to the papaya mosaic virus (PapMV) vaccine platform

With the emergence of highly virulent influenza viruses and the consequent risk of pandemics, new approaches to designing universal influenza vaccines are urgently needed. In this report, we demonstrate the potential of using a papaya mosaic virus (PapMV) platform carrying the universal M2e influenza epitope (PapMV-CP-M2e) as a candidate flu vaccine. We show that PapMV-CP-M2e virus-like particles (VLPs) can induce production in mice of anti-M2e antibodies that can recognize influenza-infected cells. PapMV-CP-M2e discs made of 20 coat protein (CP) subunits were shown to be poorly immunogenic compared to PapMV-CP-M2e VLPs composed of several hundred CP subunits. We also show that addition of either alum or PapMV-CP VLPs as adjuvant dramatically increased the immunogenicity of PapMV-CP-M2e-containing vaccine, and led to 100% protection against a challenge of 4LD(50) with the WSN/33 strain. These results show, for the first time, the potential of a recombinant plant virus protein to serve as both peptide delivery system and adjuvant in the crucial field of influenza vaccine development.

Household Transmission of the 2009 Pandemic A/H1N1 Influenza Virus: Elevated Laboratory-Confirmed Secondary Attack Rates and Evidence of Asymptomatic Infections

BACKGROUND Characterizing household transmission of the 2009 pandemic A/H1N1 influenza virus (pH1N1) is critical for the design of effective public health measures to mitigate spread. Our objectives were to estimate the secondary attack rates (SARs), the proportion of asymptomatic infections, and risk factors for pH1N1 transmission within households on the basis of active clinical follow-up and laboratory-confirmed outcomes. METHODS We conducted a prospective observational study during the period May-July 2009 (ie, during the first wave of the pH1N1 pandemic) in Quebec City, Canada. We assessed pH1N1 transmission in 42 households (including 43 primary case patients and 119 contacts). Clinical data were prospectively collected during serial household visits. Secondary case patients were identified by clinical criteria and laboratory diagnostic tests, including serological and molecular methods. RESULTS We identified 53 laboratory-confirmed secondary case patients with pH1N1 virus infection, for an SAR of 45% (95% confidence interval [CI], 35.6%-53.5%). Thirty-four (81%) of the households had ≥1 confirmed secondary case patient. The mean serial interval between onset of primary and confirmed secondary cases was 3.9 days (median interval, 3 days). Influenza-like illness (fever and cough or sore throat) developed in 29% (95% CI, 20.5%-36.7%) of household contacts. Five (9.4%) of secondary case patients were asymptomatic. Young children (<7 years of age) were at highest risk of developing laboratory-confirmed influenza-like illness. Primary case patients with both diarrhea and vomiting were the most likely to transmit pH1N1. CONCLUSION Household transmission of pH1N1 may be substantially greater than previously estimated, especially in association with clinical presentations that include gastrointestinal complaints. Approximately 10% of pH1N1 infections acquired in the household may be asymptomatic.

Effect of the Neuraminidase Mutation H274Y Conferring Resistance to Oseltamivir on the Replicative Capacity and Virulence of Old and Recent Human Influenza A(H1N1) Viruses

BACKGROUND The viral fitness of neuraminidase inhibitor (NAI)-resistant influenza viruses is believed to be impaired. Unexpectedly, an oseltamivir-resistant A(H1N1) variant containing the H274Y neuraminidase (NA) mutation recently disseminated worldwide, suggesting that the replication and virulence properties of this mutant virus were not compromised. METHODS In vitro replicative capacities were determined for old (A/WSN/33, A/Mississipi/3/01, A/New Caledonia/20/99, and A/Solomon Islands/03/06) and recent (A/Brisbane/59/2007-like) influenza A(H1N1) viruses either harboring or not harboring the H274Y NA mutation. Ferrets were infected with the A/Brisbane/59/2007-like wild-type (WT) isolate and its H274Y NA variant. RESULTS Old A(H1N1) WT viruses grew at higher titers than did the A/Brisbane/59/2007-like viruses in vitro. The H274Y mutation was associated with reduced viral plaque areas in cells infected with A/WSN/33 and A/Mississippi/3/01, whereas the 2 A/Brisbane/59/2007-like isolates showed similar plaque sizes. In ferrets, the pyrexic response induced by the A/Brisbane/59/2007-like H274Y mutant was significantly higher than that induced by the WT isolate. Nasal wash viral titers were significantly greater for the mutant isolate on day 2 after inoculation, whereas the 2 viruses showed similar titers between days 3 and 7 after inoculation. CONCLUSIONS The viral fitness of the recent A/Brisbane/59/2007-like H274Y variant is not impaired, consistent with its global dissemination. These results reinforce the need for new antiviral strategies.

Oseltamivir-Resistant Pandemic A/H1N1 Virus Is as Virulent as Its Wild-Type Counterpart in Mice and Ferrets

The neuraminidase inhibitor oseltamivir is currently used for treatment of patients infected with the pandemic A/H1N1 (pH1N1) influenza virus, although drug-resistant mutants can emerge rapidly and possibly be transmitted. We describe the characteristics of a pair of oseltamivir-resistant and oseltamivir-susceptible pH1N1 clinical isolates that differed by a single change (H274Y) in the neuraminidase protein. Viral fitness of pH1N1 isolates was assessed in vitro by determining replication kinetics in MDCK α2,6 cells and in vivo by performing experimental infections of BALB/c mice and ferrets. Despite slightly reduced propagation of the mutant isolate in vitro during the first 24 h, the wild-type (WT) and mutant resistant viruses induced similar maximum weight loss in mice and ferrets with an identical pyrexic response in ferrets (AUC of 233.9 and 233.2, P = 0.5156). Similarly, comparable titers were obtained for the WT and the mutant strains on days 1, 3, 6 and 9 post-infection in mouse lungs and on days 1–7 in ferret nasal washes. A more important perivascular (day 6) and pleural (days 6 and 12) inflammation was noted in the lungs of mice infected with the H274Y mutant, which correlated with increased pulmonary levels of IL-6 and KC. Such increased levels of IL-6 were also observed in lymph nodes of ferrets infected with the mutant strain. Furthermore, the H274Y mutant strain was transmitted to ferrets. In conclusion, viral fitness of the H274Y pH1N1 isolate is not substantially altered and has the potential to induce severe disease and to disseminate.

H5N1 vaccines in humans

The spread of highly pathogenic avian H5N1 influenza viruses since 1997 and their virulence for poultry and humans has raised concerns about their potential to cause an influenza pandemic. Vaccines offer the most viable means to combat a pandemic threat. However, it will be a challenge to produce, distribute and implement a new vaccine if a pandemic spreads rapidly. Therefore, efforts are being undertaken to develop pandemic vaccines that use less antigen and induce cross-protective and long-lasting responses, that can be administered as soon as a pandemic is declared or possibly even before, in order to prime the population and allow for a rapid and protective antibody response. In the last few years, several vaccine manufacturers have developed candidate pandemic and pre-pandemic vaccines, based on reverse genetics and have improved the immunogenicity by formulating these vaccines with different adjuvants. Some of the important and consistent observations from clinical studies with H5N1 vaccines are as follows: two doses of inactivated vaccine are generally necessary to elicit the level of immunity required to meet licensure criteria, less antigen can be used if an oil-in-water adjuvant is included, in general antibody titers decline rapidly but can be boosted with additional doses of vaccine and if high titers of antibody are elicited, cross-reactivity against other clades is observed. Prime-boost strategies elicit a more robust immune response. In this review, we discuss data from clinical trials with a variety of H5N1 influenza vaccines. We also describe studies conducted in animal models to explore the possibility of reassortment between pandemic live attenuated vaccine candidates and seasonal influenza viruses, since this is an important consideration for the use of live vaccines in a pandemic setting.

Development of a High-Yield Live Attenuated H7N9 Influenza Virus Vaccine That Provides Protection against Homologous and Heterologous H7 Wild-Type Viruses in Ferrets

ABSTRACT Live attenuated H7N9 influenza vaccine viruses that possess the hemagglutinin (HA) and neuraminidase (NA) gene segments from the newly emerged wild-type (wt) A/Anhui/1/2013 (H7N9) and six internal protein gene segments from the cold-adapted influenza virus A/Ann Arbor/6/60 (AA ca) were generated by reverse genetics. The reassortant virus containing the original wt A/Anhui/1/2013 HA and NA sequences replicated poorly in eggs. Multiple variants with amino acid substitutions in the HA head domain that improved viral growth were identified by viral passage in eggs and MDCK cells. The selected vaccine virus containing two amino acid changes (N133D/G198E) in the HA improved viral titer by more than 10-fold (reached a titer of 108.6 fluorescent focus units/ml) without affecting viral antigenicity. Introduction of these amino acid changes into an H7N9 PR8 reassortant virus also significantly improved viral titers and HA protein yield in eggs. The H7N9 ca vaccine virus was immunogenic in ferrets. A single dose of vaccine conferred complete protection of ferrets from homologous wt A/Anhui/1/2013 (H7N9) and nearly complete protection from heterologous wt A/Netherlands/219/2003 (H7N7) challenge infection. Therefore, this H7N9 live attenuated influenza vaccine (LAIV) candidate has been selected for vaccine manufacture and clinical evaluation to protect humans from wt H7N9 virus infection. IMPORTANCE In response to the recent avian H7N9 influenza virus infection in humans, we developed a live attenuated H7N9 influenza vaccine (LAIV) with two amino acid substitutions in the viral HA protein that improved vaccine yield by 10-fold in chicken embryonated eggs, the substrate for vaccine manufacture. The two amino acids also improved the antigen yield for inactivated H7N9 vaccines, demonstrating that this finding could great facilitate the efficiency of H7N9 vaccine manufacture. The candidate H7N9 LAIV was immunogenic and protected ferrets against homologous and heterologous wild-type H7 virus challenge, making it suitable for use in protecting humans from H7 infection.

A Novel Neuraminidase Deletion Mutation Conferring Resistance to Oseltamivir in Clinical Influenza A/H3N2 Virus

Neuraminidase (NA) mutations responsible for influenza resistance to oseltamivir vary according to the NA subtype; in influenza A/H3N2 viruses, NA-gene mutations occur predominantly at codons E119 and R292. In an oseltamivir-resistant influenza A/H3N2 virus isolated from an immunocompromised child after 107 days of cumulative treatment, the NA gene contained 3 aa substitutions (N146K, S219T, and A272V) and a 4-aa deletion (Del245-248); reversion mutation experiments using recombinant NA proteins determined that the deletion was the sole change responsible for resistance to oseltamivir. This study highlights the diversity of mechanisms of resistance to oseltamivir in clinical settings, reinforcing the need for novel anti-influenza strategies.

Infections by human coronavirus-NL in hospitalized children.

Background: A new human coronavirus (HCoV), HCoV-NL, was recently reported for Dutch patients with acute respiratory tract infections (ARTI). Little information is available on the incidence, clinical manifestations and epidemiologic features of HCoV-NL infections. Methods: We performed a prospective study of symptomatic (case subjects with ARTI) and asymptomatic (control subjects undergoing elective surgery) children, ≤3 years of age, hospitalized in Canada during 2 winter seasons (2001–2003), to look at the prevalence of respiratory viruses. Reverse transcription-PCR assays were used to retrospectively detect HCoV-NL and to correlate its presence with clinical symptoms. Results: HCoV-NL was detected in nasopharyngeal aspirates from 3.0% of young children (12 of 396 children) hospitalized for treatment of ARTI (case subjects), compared with 1.7% of asymptomatic control subjects (3 of 177 children) (P = 0.6). Nine (75.0%) of the symptomatic children had mixed viral infections. The mean age and mean duration of hospitalization of case subjects were 10.1 months and 4.9 days, respectively. Final diagnoses consisted of bronchiolitis or bronchitis (9 of 12 cases), pneumonitis (1 of 12 cases) and upper respiratory tract infections (2 of 12 cases), although 2 of 3 subjects with single HCoV-NL infections had upper respiratory tract infections only. Sequence analysis of the 1a and spike genes revealed that multiple HCoV-NL strains circulated in the same geographical area in each of the 2 winter seasons. Variability was more pronounced for the spike gene, with 2 clusters of strains. Conclusions: HCoV-NL was not a major respiratory pathogen in Canada during our study, as shown by its low detection rate in hospitalized children with ARTI, coupled with the high frequency of additional pathogens and its occasional detection in healthy children.

Immunogenicity and tolerability of an inactivated and adjuvanted pandemic H1N1 influenza vaccine, in HIV-1-infected patients.

We evaluated the efficacy and tolerability of a single dose of the split virion AS03-adjuvanted pandemic H1N1 influenza vaccine (A/California/7/2009) in 84 HIV-1 infected individuals. Antibody titers were determined by hemagglutination inhibition assay and by microneutralization. Vaccine was well tolerated. At 21 days post vaccination, 56 (67%) patients had seroconverted. There was no correlation between baseline CD4 cell count (p=0.539) or HIV viral load (p=0.381) and immune response. Other vaccine strategies should be evaluated in this HIV population, to improve response rates.