Understanding the Impact of Resistance to Influenza Antivirals.

Influenza poses a significant burden on society and health care systems. Although antivirals are an integral tool in effective influenza management, the potential for the emergence of antiviral-resistant viruses can lead to uncertainty and hesitation among front-line prescribers and policy makers. Here, we provide an overview of influenza antiviral resistance in context, exploring the key concepts underlying its development and clinical impact. Due to the acute nature of influenza in immunocompetent patients, resistant viruses that develop during antiviral treatment of a single patient ("treatment-emergent resistance") are usually cleared in a relatively short time, with no impact on future antiviral efficacy. In addition, although available data are limited by small numbers of patients, they show that antiviral treatment still provides clinical benefit to the patient within whom resistance emerges. In contrast, the sustained community transmission of resistant variants in the absence of treatment ("acquired resistance") is of greater concern and can potentially render front-line antivirals ineffective. Importantly, however, resistant viruses are usually associated with reduced fitness such that their widespread transmission is relatively rare. Influenza antivirals are an essential part of effective influenza management due to their ability to reduce the risk of complications and death in infected patients. Although antiviral resistance should be taken seriously and requires continuous careful monitoring, it is not comparable to antibiotic resistance in bacteria, which can become permanent and widespread, with far-reaching medical consequences. The benefits of antiviral treatment far outweigh concerns of potential resistance, which in the vast majority of cases does not have a significant clinical impact.

Towards a universal influenza vaccine: volunteer virus challenge studies in quarantine to speed the development and subsequent licensing.

There are now more than 5 experimental vaccine formulations which induce T and B cell immunity towards the internally situated virus proteins matrix (M1 and M2e) and nucleoprotein (NP), and towards stem and stalk regions of the HA which have a shared antigenic structure amongst many of the 17 influenza A virus sub types. Such 'universal vaccines' could be used, at least in theory, as a prophylactic stockpile vaccine for newly emerged epidemic and novel pandemic influenza A viruses or as a supplement to conventional HA/NA vaccines. My own laboratory has approached the problem from the clinical viewpoint by identifying CD4(+) cells which are present in influenza infected volunteers who resist influenza infection. We have established precisely which peptides in M and NP proteins react with these immune CD4 cells. These experimental vaccines induce immunity in animal models but with a single exception no data have been published on protection against influenza virus infection in humans. The efficacy of the latter vaccine is based on vaccinia virus (MVA) as a carrier and was analyzed in a quarantine unit. Given the absence of induced HI antibody in the new universal vaccines a possible licensing strategy is a virus challenge model in quarantine whereby healthy volunteers can be immunized with the new vaccine and thereafter deliberately infected and clinical signs recorded alongside quantities of virus excreted and compared with unvaccinated controls.

Vaccines from the Spanish Influenza as a firm foundation for new developments.

In 1914, the concept of a prophylactic vaccine, administered to a person before the disease had been contracted, was still controversial. Nevertheless, Almroth Wright tested new pneumococcus vaccines in South Africa, where the incidence of bacterial pneumonia was high amongst workers in the gold mines. He established the use of clinical trials, using around ten thousand workers, both in vaccinated and unvaccinated groups. The two groups were not matched to modern standards. Also, of course, those workers in the control unvaccinated group could not be protected: but some considered a prophylactic vaccine would exacerbate the disease. The vaccines were manufactured to contain a range of pneumococci from different clinical samples, in a serious attempt to match the microbes in the vaccine to the field bacteria. Deaths were averted by the vaccine; and side effects were noted to be minimal. Reexamination of pathology samples from the Spanish Influenza Pandemic showed quite clearly the contribution of pneumococci and streptococci to the mortality of over fifty million people in 1918-1919. The microbe causing this Pandemic was isolated in 1933, and was shown to be a true virus; this finding initiated a huge expanse and interest in influenza virus vaccines, both killed and live. A chance discovery allowed the purification of Influenza M and NP proteins then permitted the production of experimental vaccines. These vaccines were formulated to induce and B and/or T cell responses to the internal proteins. Several of these Universal Influenza Vaccines have been tested in quarantine, and have now reached Phase III trials in the community.

A possible European origin of the Spanish influenza and the first attempts to reduce mortality to combat superinfecting bacteria: an opinion from a virologist and a military historian.

When we reconsider the virology and history of the Spanish Influenza Pandemic, the science of 2018 provides us with tools which did not exist at the time. Two such tools come to mind. The first lies in the field of 'gain of function' experiments. A potential pandemic virus, such as influenza A (H5N1), can be deliberately mutated in the laboratory in order to change its virulence and spreadability. Key mutations can then be identified. A second tool lies in phylogenetics, combined with molecular clock analysis. It shows that the 1918 pandemic virus first emerged in the years 1915-1916. We have revisited the literature published in Europe and the United States, and the notes left by physicians who lived at the time. In this, we have followed the words of the late Alfred Crosby: who wrote that "contemporary documentary evidence from qualified physicians" is the key to understanding where and how the first outbreaks occurred. In our view, the scientists working in Europe fulfill Crosby's requirement for contemporary evidence of origin. Elsewhere, Crosby also suggested that "the physicians of 1918 were participants in the greatest failure of medical science in the twentieth century". Ours is a different approach. We point to individual pathologists in the United States and in France, who strove to construct the first universal vaccines against influenza. Their efforts were not misdirected, because the ultimate cause of death in nearly all cases flowed from superinfections with respiratory bacteria.

Lack of evidence for complete resistance of peripheral blood mononuclear cells to HIV-1 and HIV-2 infection.

There are reports that not all individuals exposed to HIV-1 become infected and the possibility exists that some individuals may be completely resistant to infection with this virus. This study aims to investigate, in vitro, whether certain peripheral blood mononuclear cells (PBMCs) are completely resistant to HIV-1 and HIV-2 infection. PBMCs obtained from 130 unrelated healthy HIV-1- and HIV-2-seronegative volunteers were infected with four different isolates of HIV-1 (H995 and MN) and HIV-2 (CBL-20 and ROD) using several multiplicities of infection. Cultures were maintained for 21 d. Virus replication was measured using the viral p24 core antigen levels in the case of HIV-1, and by reverse transcriptase (RT) activity in the case of HIV-2, at 5, 14, and 21 d post-infection. Marked variations were observed among PBMCs from individual donors with regard to replication rates for HIV-1 and HIV-2. None of the PBMCs from any single donor was shown to have zero viral replication rates for all four HIV isolates tested. However, PBMCs from some individuals were shown to have either very low or very high viral replication rates when infected with one or more virus isolates. Our results clearly distinguished three groups of PBMCs with varying degrees of viral replication for both HIV-1 and HIV-2 infection in vitro: (a) those with high viral replication rates, (b) those with moderate viral replication rates, and (c) those with low viral replication rates. Our data indicate that although none of the PBMCs tested were shown to be completely resistant to in vitro HIV-1 and HIV-2 infection, partial resistance to infection was seen for some donor samples.

Unanswered questions about the 1918 influenza pandemic: origin, pathology, and the virus itself.

The influenza epidemic of 1918 represented the greatest failure of medical science in the 20th century. Fortunately, research throughout subsequent years has been making amends. Some studies have applied RT-PCR to the tissue samples from that time, whereas others have reconstructed the pathogen in its virulent state. But the resurrection of the 1918 influenza virus leaves questions unanswered: although more virulent than contemporary H1N1 epidemic viruses in animal models, this increased virulence of the 1918 influenza virus is not sufficient to have been the sole cause of the high mortality rates recorded in humans during the epidemic. Thus, other hypotheses have been investigated. The immune history of the different age groups exposed at the time to the pandemic virus could be a factor, and the notion of original antigenic sin provides an explanation for the unusual pattern of deaths. The presence, or absence, of a cytokine storm in the lungs of young adults might also be involved. The time and location that the 1918 influenza pandemic first emerged from its avian reservoir is contentious, with arguments for China, Europe, and the USA, at various dates. Novel vaccines were tested during 1918, which are the precursors of the universal influenza vaccines that might offer protection in a future pandemic.

Functional and antigenic analyses of the 1918 influenza virus haemagglutinin using a recombinant vaccinia virus expression system.

The influenza pandemic of 1918 caused unprecedented levels of morbidity and mortality in its 12-month period of circulation around the globe. The haemagglutinin molecule has been shown to affect the pathogenicity of some subtypes of influenza A viruses. Using a recombinant vaccinia system that allowed expression of the 1918 influenza haemagglutinin, we performed functional assays to assess the glycoprotein's involvement in determining the high pathogenicity of the 1918 virus. We show that in respect of expression levels, proteolytic processing, receptor-binding, membrane fusion and antigenic properties, the haemagglutinin of the 1918 virus is unremarkable when compared with the haemagglutinins of other 'early' H1 influenza viruses. This suggests that whilst the 1918 haemagglutinin, as a new/novel antigen in the human population, was responsible for the influenza pandemic its functions per se were not responsible for the high mortality and acute symptoms experienced by patients infected with the 1918 influenza virus.

An Intranasal Proteosome-Adjuvanted Trivalent Influenza Vaccine Is Safe, Immunogenic & Efficacious in the Human Viral Influenza Challenge Model. Serum IgG & Mucosal IgA Are Important Correlates of Protection against Illness Associated with Infection.

INTRODUCTION: A Proteosome-adjuvanted trivalent inactivated influenza vaccine (P-TIV) administered intra-nasally was shown to be safe, well tolerated and immunogenic in both systemic and mucosal compartments, and effective at preventing illness associated with evidence of influenza infection. METHODS: In two separate studies using the human viral challenge model, subjects were selected to be immunologically naive to A/Panama/2007/1999 (H3N2) virus and then dosed via nasal spray with one of three regimens of P-TIV or placebo. One or two doses, 15 µg or 30 µg, were given either once only or twice 14 days apart (1 x 30 µg, 2 x 30 µg, 2 x 15 µg) and subjects were challenged with A/Panama/2007/1999 (H3N2) virus. Immune responses to the vaccine antigens were measured by haemagglutination inhibition assay (HAI) and nasal wash secretory IgA (sIgA) antibodies. RESULTS: Vaccine reactogenicity was mild, predictable and generally consistent with earlier Phase I studies with this vaccine. Seroconversion to A/Panama/2007/1999 (H3N2), following vaccination but prior to challenge, occurred in 57% to 77% of subjects in active dosing groups and 2% of placebo subjects. The greatest relative rise in sIgA, following vaccination but prior to challenge, was observed in groups that received 2 doses. CONCLUSION: Intranasal vaccination significantly protected against influenza (as defined by influenza symptoms combined with A/Panama seroconversion) following challenge with A/Panama/2007/1999 (H3N2). When data were pooled from both studies, efficacy ranged from 58% to 82% in active dosing groups for any influenza symptoms with seroconversion, 67% to 85% for systemic or lower respiratory illness and seroconversion, and 65% to 100% for febrile illness and seroconversion. The two dose regimen was found to be superior to the single dose regimen. In this study, protection against illness associated with evidence of influenza infection (evidence determined by seroconversion) following challenge with virus, significantly correlated with pre-challenge HAI titres (p = 0.0003) and mucosal sIgA (p≤0.0001) individually, and HAI (p = 0.028) and sIgA (p = 0.0014) together. HAI and sIgA levels were inversely related to rates of illness. TRIAL REGISTRATION: ClinicalTrials.gov NCT02522754.

New antiviral drugs, vaccines and classic public health interventions against SARS coronavirus.

Severe acute respiratory syndrome (SARS) is caused by one of two recently discovered coronaviruses. The virus is emergent from South East (SE) Asian mammals: either the civet cat, a related species or a rat species. The virus has a long incubation period and low reproduction number (R0 value) and hence the first outbreak in 2004 was controlled by hygiene and quarantine. However, the healthcare system was compromised and the economic cost was extremely high. Fortunately, the virus is easily cultivated in Vero E6 cells and therefore the search for new antivirals and vaccines was initiated within weeks of the discovery of the virus using classic techniques of cell culture and electron microscopy. Molecular diagnostics facilitated rapid and accurate diagnosis, a key factor in containing the outbreak. The broad-spectrum molecule ribavirin was used in SE Asia in infected patients alongside corticosteroids. In retrospect, many patients survived due to careful nursing. The only currently accepted intervention is interferon. Coronavirus replicon systems should facilitate rapid screening of new inhibitors and the complex mechanism of viral replication will ensure that drugs are developed against at least five molecular targets, in particular the viral protease.

A throat lozenge containing amyl meta cresol and dichlorobenzyl alcohol has a direct virucidal effect on respiratory syncytial virus, influenza A and SARS-CoV.

A potent virucidal mixture containing amyl metacresol and dichlorobenzyl alcohol at low pH inactivated enveloped respiratory viruses influenza A, respiratory synctial virus (RSV) and severe acute respiratory syndrome coronavirus (SARS-CoV) but not viruses with icosahedral symmetry, such as adenoviruses or rhinoviruses. A titre of approximately 3.5 log10 TCID50 was reduced to below the level of detection within two minutes. Electron microscopy of purified influenza A virus showed extensive clumping and morphological changes in spike configuration after contact with the virucidal mixture, but no overt destruction of the viral membrane. We conclude that, formulated as a lozenge, the mixture could have significant effects in reducing the infectivity of certain infectious viruses in the throat and presumably in cough droplets, thus reducing, theoretically, opportunities for person-to-person transmission.

The prevalence of myocarditis and skeletal muscle injury during acute viral infection in adults: measurement of cardiac troponins I and T in 152 patients with acute influenza infection.

BACKGROUND: Current literature suggests that myocarditis is a common event during influenza infection, occurring with a prevalence rate of up to 10%, but these studies have relied on relatively nonspecific techniques of varying sensitivities for the detection of myocyte injury. Using measurement of cardiac troponins I and T, this study sought to determine the prevalence of myocarditis in a large unselected cohort of patients with serologically confirmed acute influenza infection. METHODS: A total of 152 subjects were recruited from 60 primary care and university health centers. Serial creatine kinase (CK), CK-MB, and cardiac troponin I and T measurements were taken on days 1, 6, and 21 following presentation. RESULTS: Creatine kinase levels were elevated (mean +/- SD levels, 830 +/- 1531 U/L; range, 181-7280 U/L) during the collection period in 18 patients (12%). Twelve (67%) of these had elevated CK levels on day 1 of presentation. Overall CK-MB levels were higher than 25 U/L in 3 patients with elevated CK readings but in no patient was the CK-MB fraction greater than 6%. Cardiac troponin I and T levels were not raised in any of the patients. CONCLUSIONS: Using more sensitive and specific markers of myocardial injury, we demonstrate that the prevalence of myocarditis during acute influenza infection is substantially lower than previously thought, whereas skeletal muscle injury is relatively common. Although we were unable to conclude that no myocardial inflammation was present, it seems likely that this complication is rare.

Immunogold electron microscopy recognizes prion protein-associated particles prepared from scrapie-infected mouse brain.

Previous studies have proposed that the disease isoform of prion protein (PrPSc) is particulate. Our purpose was to search by electron microscopy (EM) for particles in fractions of density gradients prepared from differentially centrifuged homogenates of scrapie-infected, normal, and null mouse brain. Only mild detergents were used during the separation process. The low-density fractions derived from scrapie-infected brain were rich in PrP. Three morphologically distinct types of particle were observed. Type 1 particles, measuring approximately 6.8 nm in mean diameter, were found in abundance in the fractions of scrapie-infected brain at the peak PrP concentrations. They were often clumped and adherent to raft-like structures. Type 2 particles, in low-density fractions from normal brain, were similar to type 1 but were smaller, with the mean diameter measuring approximately 5.3 nm. Type 3 particles from null brain differed morphologically from types 1 and 2 and were not clumped. The low density of the particles indicated a lipid component, which was confirmed by lipid analysis. Immunogold EM using Mab 6H4 labeled a portion of the particles from scrapie mouse brain, but not those from normal or null brain. Dimensions of PrP suggest that the labeled particles carry a PrPSc dimer per particle.

New millennium antivirals against pandemic and epidemic influenza: the neuraminidase inhibitors.

The mushroom shaped outer spike protein of influenza, neuraminidase, was first discovered nearly 60 years ago. Its importance in viral replication was soon recognised both at the point of viral release from the cell and also enabling passage of virus through nasal fluid to reach the cell. The enzyme active site was identified by x-ray crystallography, allowing an atomic study of interaction of enzyme with the sialic acid substrate. Analogues could then be identified and synthesized and became a focused target for antivirals. With the current threat of bioterrorism and the potential for the emergence of a new pandemic strain in the near future, efforts are underway to develop more potent second-generation anti-neuraminidase inhibitors with enhanced protective and therapeutic effects. Here we review older and newer neuraminidase inhibitors and the role that they will play in the fight against influenza in its epidemic and pandemic face.

Influenza A: a threatening virus with two faces.

In 1918, a great 'forgotten plague' wiped out fifty million people around the world - many of them young and healthy. The killer disease was flu. What was it that made this outbreak so deadly and would we be prepared if another pandemic struck?

Intranasal H5N1 vaccines, adjuvanted with chitosan derivatives, protect ferrets against highly pathogenic influenza intranasal and intratracheal challenge.

We investigated the protective efficacy of two intranasal chitosan (CSN and TM-CSN) adjuvanted H5N1 Influenza vaccines against highly pathogenic avian Influenza (HPAI) intratracheal and intranasal challenge in a ferret model. Six groups of 6 ferrets were intranasally vaccinated twice, 21 days apart, with either placebo, antigen alone, CSN adjuvanted antigen, or TM-CSN adjuvanted antigen. Homologous and intra-subtypic antibody cross-reacting responses were assessed. Ferrets were inoculated intratracheally (all treatments) or intranasally (CSN adjuvanted and placebo treatments only) with clade 1 HPAI A/Vietnam/1194/2004 (H5N1) virus 28 days after the second vaccination and subsequently monitored for morbidity and mortality outcomes. Clinical signs were assessed and nasal as well as throat swabs were taken daily for virology. Samples of lung tissue, nasal turbinates, brain, and olfactory bulb were analysed for the presence of virus and examined for histolopathological findings. In contrast to animals vaccinated with antigen alone, the CSN and TM-CSN adjuvanted vaccines induced high levels of antibodies, protected ferrets from death, reduced viral replication and abrogated disease after intratracheal challenge, and in the case of CSN after intranasal challenge. In particular, the TM-CSN adjuvanted vaccine was highly effective at eliciting protective immunity from intratracheal challenge; serologically, protective titres were demonstrable after one vaccination. The 2-dose schedule with TM-CSN vaccine also induced cross-reactive antibodies to clade 2.1 and 2.2 H5N1 viruses. Furthermore ferrets immunised with TM-CSN had no detectable virus in the respiratory tract or brain, whereas there were signs of virus in the throat and lungs, albeit at significantly reduced levels, in CSN vaccinated animals. This study demonstrated for the first time that CSN and in particular TM-CSN adjuvanted intranasal vaccines have the potential to protect against significant mortality and morbidity arising from infection with HPAI H5N1 virus.

Correlation between human leukocyte antigen class II alleles and HAI titers detected post-influenza vaccination.

Influenza is a major cause of morbidity and mortality. Despite vaccination, many elderly recipients do not develop a protective antibody response. To determine whether Human Leukocyte Antigen (HLA) alleles modulate seroprotection to influenza, a cohort of HLA class II-typed high-risk vaccine recipients was investigated. Haemagglutinin inhibition (HAI) titres were measured 14-40 days post-subunit vaccination. Seroprotection was defined as HAI titres reaching 40 or greater for all three vaccine strains. HLA-DRB1*04∶01 and HLA-DPB1*04∶01 alleles were detected at higher frequencies in seroprotected compared with non-seroprotected individuals. Thus, the presence of certain HLA class II alleles may determine the magnitude of antibody responses to influenza vaccination.

Comparing influenza and RSV viral and disease dynamics in experimentally infected adults predicts clinical effectiveness of RSV antivirals.

BACKGROUND: Antivirals reduce influenza viral replication and illness measures, particularly if initiated early, within 48 h of symptom onset. Whether experimental antivirals that reduce respiratory syncytial virus (RSV) load would also reduce disease is unknown. This study compares viral and disease dynamics in humans experimentally infected with influenza or RSV. METHODS: Clinical strains of RSV-A and influenza A were inoculated intranasally into 20 and 17 healthy volunteers, respectively, on day 0. Symptom scores and nasal washes were performed twice daily, and daily mucus weights were collected. Viral loads in nasal washes were quantified by culture (plaque assay in HEp-2 cells for RSV and by end point dilution in Madin-Darby canine kidney cells for influenza). RESULTS: After influenza inoculation, influenza viral load and illness markers increased simultaneously until day 2. Within individual subjects, peak influenza load occurred 0.4 days (95% CI -0.4, 1.3) before peak symptoms. Influenza viral load and disease declined thereafter. After RSV inoculation, a longer incubation period occurred prior to viral detection and symptom onset. RSV load and disease increased together until day 5. Within individual subjects, peak RSV loads occurred 0.2 days (95% CI -0.7, 1.05) before peak symptoms, after which both illness measures and viral load declined together. CONCLUSIONS: Viral and disease dynamics in experimental human infections suggest that reducing RSV load, if timed similarly to clinically-effective influenza antivirals, might be expected to have a similar or greater window of opportunity for reducing clinical RSV disease.

Clinical, scientific and ethnographic studies of influenza in quarantine.

From the time of the Spanish influenza pandemic in 1918 to the present seclusion of volunteers in quarantine units, either modified hotels, Phase I units or specially constructed clinics, have been key in investigating new vaccines and antivirals. Carefully selected healthy, young volunteers undergo a 10-12-day sojourn under intense medical supervision. Clinical sampling includes nasal and throat washes for virus recovery, blood for clinical chemistry, analysis of B- and T-cell response and, more recently, analysis of human genes responding to infection. The authors' studies are resulting in new developments of universal influenza vaccines that could stimulate and prime CD4 and CD8 cells to shared epitopes in all influenza A viruses. Ethnographic study has noted an absence of quarantine stress in the volunteers for the first time.