Deep sequencing of 2009 influenza A/H1N1 virus isolated from volunteer human challenge study participants and natural infections.

Nasal wash samples from 15 human volunteers challenged with GMP manufactured influenza A/California/04/2009(H1N1) and from 5 naturally infected influenza patients of the 2009 pandemic were deep sequenced using viral targeted hybridization enrichment. Ten single nucleotide polymorphism (SNP) positions were found in the challenge virus. Some of the nonsynonymous changes in the inoculant virus were maintained in some challenge participants, but not in others, indicating that virus is evolving away from the Vero cell adapted inoculant, for example SNPs in the neuraminidase. Many SNP sites in challenge patients and naturally infected patients were found, many not identified previously. The SNPs identified, and phylogenetic analyses, showed that intrahost evolution of the virus are different in challenge participants and naturally infected patients. This study, using hybridization enrichment without PCR, provided an accurate and unbiased assessment of differential intrahost viral evolution from a uniform influenza inoculant in humans and comparison to naturally infected patients.

Differential Effects of Influenza Virus NA, HA Head, and HA Stalk Antibodies on Peripheral Blood Leukocyte Gene Expression during Human Infection.

In this study, we examined the relationships between anti-influenza virus serum antibody titers, clinical disease, and peripheral blood leukocyte (PBL) global gene expression during presymptomatic, acute, and convalescent illness in 83 participants infected with 2009 pandemic H1N1 virus in a human influenza challenge model. Using traditional statistical and logistic regression modeling approaches, profiles of differentially expressed genes that correlated with active viral shedding, predicted length of viral shedding, and predicted illness severity were identified. These analyses further demonstrated that challenge participants fell into three peripheral blood leukocyte gene expression phenotypes that significantly correlated with different clinical outcomes and prechallenge serum titers of antibodies specific for the viral neuraminidase, hemagglutinin head, and hemagglutinin stalk. Higher prechallenge serum antibody titers were inversely correlated with leukocyte responsiveness in participants with active disease and could mask expression of peripheral blood markers of clinical disease in some participants, including viral shedding and symptom severity. Consequently, preexisting anti-influenza antibodies may modulate PBL gene expression, and this must be taken into consideration in the development and interpretation of peripheral blood diagnostic and prognostic assays of influenza infection.IMPORTANCE Influenza A viruses are significant human pathogens that caused 83,000 deaths in the United States during 2017 to 2018, and there is need to understand the molecular correlates of illness and to identify prognostic markers of viral infection, symptom severity, and disease course. Preexisting antibodies against viral neuraminidase (NA) and hemagglutinin (HA) proteins play a critical role in lessening disease severity. We performed global gene expression profiling of peripheral blood leukocytes collected during acute and convalescent phases from a large cohort of people infected with A/H1N1pdm virus. Using statistical and machine-learning approaches, populations of genes were identified early in infection that correlated with active viral shedding, predicted length of shedding, or disease severity. Finally, these gene expression responses were differentially affected by increased levels of preexisting influenza antibodies, which could mask detection of these markers of contagiousness and disease severity in people with active clinical disease.

A Dose-finding Study of a Wild-type Influenza A(H3N2) Virus in a Healthy Volunteer Human Challenge Model.

BACKGROUND: The development of vaccines and therapeutics has relied on healthy volunteer influenza challenge studies. A validated human infection model with wild-type A(H1N1)pdm09 was reported previously. Our objective was to characterize a wild-type influenza A/Bethesda/MM1/H3N2 challenge virus in healthy volunteers. METHODS: Participants received a single dose of a cell-based, reverse-genetics, Good Manufacturing Practices-produced wild-type influenza A(H3N2)2011 virus intranasally and were isolated at the National Institutes of Health Clinical Center for ≥9 days. Dose escalation was performed from 104 to 107 TCID50 (50% tissue culture infectious dose). Viral shedding and clinical disease were evaluated daily. RESULTS: Of 37 participants challenged, 16 (43%) had viral shedding and 27 (73%) developed symptoms, with 12 (32%) participants experiencing mild to moderate influenza disease (MMID), defined as shedding and symptoms. Only participants receiving 106 and 107 TCID50 experienced MMID at 44% and 40%, respectively. Symptom severity peaked on day 3, whereas most viral shedding occurred 1-2 days after challenge. Only 10 (29%) participants had a ≥4-fold rise in hemagglutination inhibition antibody titer after challenge. CONCLUSIONS: The A/Bethesda/MM1/H3N2 challenge virus safely induced MMID in healthy volunteers, but caused less MMID than the A(H1N1)pdm09 challenge virus even at the highest dose. There was less detection of shedding though the incidence of symptoms was similar to A(H1N1)pdm09. Fewer serum anti-hemagglutinin (HA) antibody responses with less MMID indicate that preexisting immunity factors other than anti-HA antibody may limit shedding in healthy volunteers. This A/Bethesda/MM1/H3N2 challenge virus can be utilized in future studies to further explore pathogenesis and immunity and to evaluate vaccine candidates. CLINICAL TRIALS REGISTRATION: NCT02594189.

Design and validation of a universal influenza virus enrichment probe set and its utility in deep sequence analysis of primary cloacal swab surveillance samples of wild birds.

Influenza virus infections in humans and animals are major public health concerns. In the current study, a set of universal influenza enrichment probes was developed to increase the sensitivity of sequence-based virus detection and characterization for all influenza viruses. This universal influenza enrichment probe set contains 46,953 120nt RNA biotin-labeled probes designed based on all available influenza viral sequences and it can be used to enrich for influenza sequences without prior knowledge of type or subtype. Marked enrichment was demonstrated in influenza A/H1N1, influenza B, and H1-to-H16 hemagglutinin plasmids spiked into human DNA and in cultured influenza A/H2N1 virus. Furthermore, enrichment effects and mixed influenza A virus infections were revealed in wild bird cloacal swab samples. Therefore, this universal influenza virus enrichment probe system can capture and enrich influenza viral sequences selectively and effectively in different samples, especially ones with degraded RNA or containing low amount of influenza RNA.

Evaluation of Preexisting Anti-Hemagglutinin Stalk Antibody as a Correlate of Protection in a Healthy Volunteer Challenge with Influenza A/H1N1pdm Virus.

Influenza virus hemagglutinin (HA) surface glycoprotein is currently the primary target of licensed influenza vaccines. Recently, broadly reactive antibodies that target the stalk region of the HA have become a major focus of current novel vaccine development. These antibodies have been observed in humans after natural infection with influenza A virus, but the data are limited. Using samples and data from the uniquely controlled setting of an influenza A/H1N1 virus human challenge study of healthy volunteers, we performed a secondary analysis that for the first time explores the role of anti-HA stalk antibody as a human correlate of protection. An anti-HA stalk antibody enzyme-linked immunosorbent assay (ELISA) was performed on samples from 65 participants challenged with a 2009 H1N1pdm virus. Pre- and postchallenge anti-HA stalk titers were then correlated with multiple outcome measures to evaluate anti-HA stalk antibody titer as a correlate of protection. Anti-HA stalk antibody titers were present before challenge and rose in response to challenge in 64% of individuals. Those individuals with higher titers at baseline were less likely to develop shedding, but not less likely to develop symptoms. Similar to the hemagglutination inhibition (HAI) titer, the baseline anti-HA stalk antibody titer did not independently predict a decrease in the severity of influenza disease, while the antineuraminidase (neuraminidase inhibition [NAI]) titer did. As a correlate of protection, the naturally occurring anti-HA stalk antibody titer is predictive of a reduction of certain aspects of disease similar to HAI titer, but the NAI titer is the only identified correlate that is an independent predictor of a reduction of all assessed influenza clinical outcome measures.IMPORTANCE This is the first study to evaluate preexisting anti-HA stalk antibodies as a predictor of protection. We use a healthy volunteer influenza challenge trial for an examination of the role such antibodies play in protection. This study demonstrates that anti-HA stalk antibodies are naturally generated in response to an infection, but there is significant variability in response. Similar to antibodies that target the HA head, baseline anti-HA stalk antibody titer is a correlate of protection in terms of reduced shedding, but it is not a predictor of reduced clinical disease or an independent predictor of disease severity. These results, in the context of the limited data available in humans, suggest that vaccines that induce anti-HA stalk antibodies could play a role in future vaccine strategies, but alone, this target may be insufficient to induce a fully protective vaccine and overcome some of the issues identified with current vaccines.

Evaluation of Antihemagglutinin and Antineuraminidase Antibodies as Correlates of Protection in an Influenza A/H1N1 Virus Healthy Human Challenge Model.

UNLABELLED: Despite long-term investment, influenza continues to be a significant worldwide problem. The cornerstone of protection remains vaccination, and approved vaccines seek to elicit a hemagglutination inhibition (HAI) titer of ≥1:40 as the primary correlate of protection. However, recent poor vaccine performance raises questions regarding the protection afforded and whether other correlates of protection should be targeted. A healthy volunteer challenge study was performed with a wild-type 2009 A(H1N1)pdm influenza A challenge virus at the NIH Clinical Center to evaluate two groups of participants with HAI titers of ≥1:40 and <1:40. The primary objective was to determine whether participants with HAI titers of ≥1:40 were less likely to develop mild to moderate influenza disease (MMID) after intranasal inoculation. HAI titers of ≥1:40 were protective against MMID but did not reduce the incidence of symptoms alone. Although the baseline HAI titer correlated with some reduction in disease severity measures, overall, the baseline NAI titer correlated more significantly with all disease severity metrics and had a stronger independent effect on outcome. This study demonstrates the importance of examining other immunological correlates of protection rather than solely HAI titers. This challenge study confirms the importance of NAI titer as a correlate and for the first time establishes that it can be an independent predictor of reduction of all aspects of influenza disease. This suggests that NAI titer may play a more significant role than previously thought and that neuraminidase immunity should be considered when studying susceptibility after vaccination and as a critical target in future influenza vaccine platforms. IMPORTANCE: This study represents the first time the current gold standard for evaluating influenza vaccines as set by the U.S. Food and Drug Administration and the European Medicines Agency Committee for Medicinal Products for Human Use, a "protective" hemagglutination inhibition (HAI) titer of ≥1:40, has been evaluated in a well-controlled healthy volunteer challenge study since the cutoff was established. We used our established wild-type influenza A healthy volunteer human challenge model to evaluate how well this antibody titer predicts a reduction in influenza virus-induced disease. We demonstrate that although higher HAI titer is predictive of some protection, there is stronger evidence to suggest that neuraminidase inhibition (NAI) titer is more predictive of protection and reduced disease. This is the first time NAI titer has been clearly identified in a controlled trial of this type to be an independent predictor of a reduction in all aspects of influenza.

Validation of the wild-type influenza A human challenge model H1N1pdMIST: an A(H1N1)pdm09 dose-finding investigational new drug study.

BACKGROUND: Healthy volunteer wild-type influenza challenge models offer a unique opportunity to evaluate multiple aspects of this important virus. Such studies have not been performed in the United States in more than a decade, limiting our capability to investigate this virus and develop countermeasures. We have completed the first ever wild-type influenza A challenge study under an Investigational New Drug application (IND). This dose-finding study will lead to further development of this model both for A(H1N1)pdm09 and other strains of influenza. METHODS: Volunteers were admitted to an isolation unit at the National Institutes of Health Clinical Center for a minimum of 9 days. A reverse genetics, cell-based, Good Manufacturing Practice (GMP)-produced, wild-type A(H1N1)pdm09 virus was administered intranasally. Escalating doses were given until a dose was reached that produced disease in a minimum of 60% of volunteers. RESULTS: An optimal dose of 10(7) tissue culture infectious dose 50 was reached that caused mild to moderate influenza disease in 69% of individuals with mean viral shedding for 4-5 days and significant rises in convalescent influenza antibody titers. Viral shedding preceded symptoms by 12-24 hours and terminated 2-3 days prior to symptom resolution, indicating that individuals may be infectious before symptom development. As expected, nasal congestion and rhinorrhea were most common, but interestingly, fever was observed in only 10% of individuals. CONCLUSIONS: This study represents the first healthy volunteer influenza challenge model using a GMP-produced wild-type virus under an IND. This unique clinical research program will facilitate future studies of influenza pathogenesis, animal model validation, and the rapid, efficient, and cost-effective evaluation of efficacy of novel vaccines and therapeutics. Clinical Trials Registration.NCT01646138.

Glycosylation, hypogammaglobulinemia, and resistance to viral infections.

Genetic defects in MOGS, the gene encoding mannosyl-oligosaccharide glucosidase (the first enzyme in the processing pathway of N-linked oligosaccharide), cause the rare congenital disorder of glycosylation type IIb (CDG-IIb), also known as MOGS-CDG. MOGS is expressed in the endoplasmic reticulum and is involved in the trimming of N-glycans. We evaluated two siblings with CDG-IIb who presented with multiple neurologic complications and a paradoxical immunologic phenotype characterized by severe hypogammaglobulinemia but limited clinical evidence of an infectious diathesis. A shortened immunoglobulin half-life was determined to be the mechanism underlying the hypogammaglobulinemia. Impaired viral replication and cellular entry may explain a decreased susceptibility to infections.

The natural history of influenza infection in the severely immunocompromised vs nonimmunocompromised hosts.

INTRODUCTION: Medical advances have led to an increase in the world's population of immunosuppressed individuals. The most severely immunocompromised patients are those who have been diagnosed with a hematologic malignancy, solid organ tumor, or who have other conditions that require immunosuppressive therapies and/or solid organ or stem cell transplants. MATERIALS AND METHODS: Medically attended patients with a positive clinical diagnosis of influenza were recruited prospectively and clinically evaluated. Nasal washes and serum were collected. Evaluation of viral shedding, nasal and serum cytokines, clinical illness, and clinical outcomes were performed to compare severely immunocompromised individuals to nonimmunocompromised individuals with influenza infection. RESULTS: Immunocompromised patients with influenza had more severe disease/complications, longer viral shedding, and more antiviral resistance while demonstrating less clinical symptoms and signs on clinical assessment. CONCLUSIONS: Immunocompromised patients are at risk for more severe or complicated influenza induced disease, which may be difficult to prevent with existing vaccines and antiviral treatments. Specific issues to consider when managing a severely immunocompromised host include the development of asymptomatic shedding, multi-drug resistance during prolonged antiviral therapy, and the potential high risk of pulmonary involvement. CLINICAL TRIALS REGISTRATION: ClinicalTrials.gov identifier NCT00533182.

In vivo evaluation of pathogenicity and transmissibility of influenza A(H1N1)pdm09 hemagglutinin receptor binding domain 222 intrahost variants isolated from a single immunocompromised patient.

The influenza A(H1N1)pdm09 virus has circulated worldwide and continued to cause complicated infections and deaths. Reports have identified an increased prevalence of the hemagglutinin receptor binding domain D222G mutation in viruses isolated from individuals who have suffered such severe infections, but this association is still unclear. Virus isolated from a nasopharyngeal wash of a severely ill immunocompromised patient at the time of diagnosis contained the D222, but isolates collected later in his course from a bronchoalveolar lavage contained primarily the G222 mutation and was mixed with a minor population of D222. These clinical isolates were compared to a G222 plaque purified virus in the ferret model. The G222 predominant clinical isolate was the most pathogenic in ferrets and developed the most diversity at the 222 amino acid position during infection, suggesting that increased diversity and not a specific polymorphism at HA 222 may be important in predicting pathogenic potential.

Multidrug-resistant 2009 pandemic influenza A(H1N1) viruses maintain fitness and transmissibility in ferrets.

BACKGROUND: The 2009 influenza A(H1N1) pandemic called attention to the limited influenza treatment options available, especially in individuals at high risk of severe disease. Neuraminidase inhibitor-resistant seasonal H1N1 viruses have demonstrated the ability to transmit well despite early data indicating that resistance reduces viral fitness. 2009 H1N1 pandemic viruses have sporadically appeared containing resistance to neuraminidase inhibitors and the adamantanes, but the ability of these viruses to replicate, transmit, and cause disease in mammalian hosts has not been fully characterized. METHODS: Two pretreatment wild-type viruses and 2 posttreatment multidrug-resistant viruses containing the neuraminidase H275Y mutation collected from immunocompromised patients infected with pandemic influenza H1N1 were tested for viral fitness, pathogenicity, and transmissibility in ferrets. RESULTS: The pretreatment wild-type viruses and posttreatment resistant viruses containing the H275Y mutation all demonstrated significant pathogenicity and equivalent viral fitness and transmissibility. CONCLUSIONS: The admantane-resistant 2009 pandemic influenza A(H1N1) virus can develop the H275Y change in the neuraminidase gene conferring resistance to both oseltamivir and peramivir without any loss in fitness, transmissibility, or pathogenicity. This suggests that the dissemination of widespread multidrug resistance similar to neuraminidase inhibitor resistance in seasonal H1N1 is a significant threat.

Assessment of route of administration and dose escalation for an adenovirus-based influenza A Virus (H5N1) vaccine in chickens.

Highly pathogenic avian influenza (HPAI) virus causes one of the most economically devastating poultry diseases. An HPAI vaccine to prevent the disease in commercial and backyard birds must be effective, safe, and inexpensive. Recently, we demonstrated the efficacy of an adenovirus-based H5N1 HPAI vaccine (Ad5.HA) in chickens. To further evaluate the potential of the Ad5.HA vaccine and its cost-effectiveness, studies to determine the minimal effective dose and optimal route of administration in chickens were performed. A dose as low as 10(7) viral particles (vp) of adenovirus-based H5N1 vaccine per chicken was sufficient to generate a robust humoral immune response, which correlated with the previously reported level of protection. Several routes of administration, including intratracheal, conjunctival, subcutaneous, and in ovo routes, were evaluated for optimal vaccine administration. However, only the subcutaneous route of immunization induced a satisfactory level of influenza virus-specific antibodies. Importantly, these studies established that the vaccine-induced immunity was cross-reactive against an H5N1 strain from a different clade, emphasizing the potential of cross-protection. Our results suggest that the Ad5.HA HPAI vaccine is safe and effective, with the potential of cross-clade protection. The ease of manufacturing and cost-effectiveness make Ad5.HA an excellent avian influenza vaccine candidate with the ability to protect poultry from HPAI virus infection. Considering the limitations of the influenza vaccine technology currently used for poultry applications, any effort aimed at overcoming those limitations is highly significant.