A pan-influenza antibody inhibiting neuraminidase via receptor mimicry.

Rapidly evolving influenza A viruses (IAVs) and influenza B viruses (IBVs) are major causes of recurrent lower respiratory tract infections. Current influenza vaccines elicit antibodies predominantly to the highly variable head region of haemagglutinin and their effectiveness is limited by viral drift1 and suboptimal immune responses2. Here we describe a neuraminidase-targeting monoclonal antibody, FNI9, that potently inhibits the enzymatic activity of all group 1 and group 2 IAVs, as well as Victoria/2/87-like, Yamagata/16/88-like and ancestral IBVs. FNI9 broadly neutralizes seasonal IAVs and IBVs, including the immune-evading H3N2 strains bearing an N-glycan at position 245, and shows synergistic activity when combined with anti-haemagglutinin stem-directed antibodies. Structural analysis reveals that D107 in the FNI9 heavy chain complementarity-determinant region 3 mimics the interaction of the sialic acid carboxyl group with the three highly conserved arginine residues (R118, R292 and R371) of the neuraminidase catalytic site. FNI9 demonstrates potent prophylactic activity against lethal IAV and IBV infections in mice. The unprecedented breadth and potency of the FNI9 monoclonal antibody supports its development for the prevention of influenza illness by seasonal and pandemic viruses.

Targeting neuraminidase: the next frontier for broadly protective influenza vaccines.

Current seasonal influenza vaccines, which mainly target hemagglutinin (HA), require annual updates due to the continuous antigenic drift of the influenza virus. Developing an influenza vaccine with increased breadth of protection will have significant public health benefits. The recent discovery of broadly protective antibodies to neuraminidase (NA) has provided important insights into developing a universal influenza vaccine, either by improving seasonal influenza vaccines or designing novel immunogens. However, further in-depth molecular characterizations of NA antibody responses are warranted to fully leverage broadly protective NA antibodies for influenza vaccine designs. Overall, we posit that focusing on NA for influenza vaccine development is synergistic with existing efforts targeting HA, and may represent a cost-effective approach to generating a broadly protective influenza vaccine.

Influenza chimeric hemagglutinin structures in complex with broadly protective antibodies to the stem and trimer interface.

Influenza virus hemagglutinin (HA) has been the primary target for influenza vaccine development. Broadly protective antibodies targeting conserved regions of the HA unlock the possibility of generating universal influenza immunity. Two group 2 influenza A chimeric HAs, cH4/3 and cH15/3, were previously designed to elicit antibodies to the conserved HA stem. Here, we show by X-ray crystallography and negative-stain electron microscopy that a broadly protective antistem antibody can stably bind to cH4/3 and cH15/3 HAs, thereby validating their potential as universal vaccine immunogens. Furthermore, flexibility was observed in the head domain of the chimeric HA structures, suggesting that antibodies could also potentially interact with the head interface epitope. Our structural and binding studies demonstrated that a broadly protective antihead trimeric interface antibody could indeed target the more open head domain of the cH15/3 HA trimer. Thus, in addition to inducing broadly protective antibodies against the conserved HA stem, chimeric HAs may also be able to elicit antibodies against the conserved trimer interface in the HA head domain, thereby increasing the vaccine efficacy.

Antiviral Approaches against Influenza Virus.

Preventing and controlling influenza virus infection remains a global public health challenge, as it causes seasonal epidemics to unexpected pandemics. These infections are responsible for high morbidity, mortality, and substantial economic impact. Vaccines are the prophylaxis mainstay in the fight against influenza. However, vaccination fails to confer complete protection due to inadequate vaccination coverages, vaccine shortages, and mismatches with circulating strains. Antivirals represent an important prophylactic and therapeutic measure to reduce influenza-associated morbidity and mortality, particularly in high-risk populations. Here, we review current FDA-approved influenza antivirals with their mechanisms of action, and different viral- and host-directed influenza antiviral approaches, including immunomodulatory interventions in clinical development. Furthermore, we also illustrate the potential utility of machine learning in developing next-generation antivirals against influenza.

Mechanistic dissection of antibody inhibition of influenza entry yields unexpected heterogeneity.

Neutralizing antibodies against influenza have generally been classified according to their recognition sites, with antibodies against the head domain of hemagglutinin thought to inhibit attachment and antibodies against the stalk region thought to inhibit fusion. Here, we report the development of a microfluidic assay to measure neutralization of viral entry that can clearly differentiate between effects on attachment and fusion. Testing multiple broadly neutralizing antibodies against the hemagglutinin stalk domain, we obtain a surprising result: some broadly neutralizing antibodies inhibit fusion only, while others inhibit both fusion and viral attachment. Antibodies binding the globular head domain primarily inhibit attachment but can also reduce the fusogenic capability of viral particles that nonetheless bind the receptor. These findings shed light on the unexpectedly heterogeneous mechanisms of antibody neutralization even within similar recognition sites. The assay we have developed also provides a tool to optimize vaccine design by permitting assessment of the elicited antibody response with greater mechanistic resolution.

A Virion-Based Combination Vaccine Protects against Influenza and SARS-CoV-2 Disease in Mice.

Vaccines targeting SARS-CoV-2 have been shown to be highly effective; however, the breadth against emerging variants and the longevity of protection remains unclear. Postimmunization boosting has been shown to be beneficial for disease protection, and as new variants continue to emerge, periodic (and perhaps annual) vaccination will likely be recommended. New seasonal influenza virus vaccines currently need to be developed every year due to continual antigenic drift, an undertaking made possible by a robust global vaccine production and distribution infrastructure. To create a seasonal combination vaccine targeting both influenza viruses and SARS-CoV-2 that is also amenable to frequent reformulation, we have developed an influenza A virus (IAV) genetic platform that allows the incorporation of an immunogenic domain of the SARS-CoV-2 spike (S) protein onto IAV particles. Vaccination with this combination vaccine elicited neutralizing antibodies and provided protection from lethal challenge with both pathogens in mice. This approach may allow the leveraging of established influenza vaccine infrastructure to generate a cost-effective and scalable seasonal vaccine solution for both influenza and coronaviruses. IMPORTANCE The rapid emergence of SARS-CoV-2 variants since the onset of the pandemic has highlighted the need for both periodic vaccination "boosts" and a platform that can be rapidly reformulated to manufacture new vaccines. In this work, we report an approach that can utilize current influenza vaccine manufacturing infrastructure to generate combination vaccines capable of protecting from both influenza virus- and SARS-CoV-2-induced disease. The production of a combined influenza/SARS-CoV-2 vaccine may represent a practical solution to boost immunity to these important respiratory viruses without the increased cost and administration burden of multiple independent vaccines.

Non-Steroidal Estrogens Inhibit Influenza Virus by Interacting with Hemagglutinin and Preventing Viral Fusion.

Influenza virus is one of the main causes of respiratory infections worldwide. Despite the availability of seasonal vaccines and antivirals, influenza virus infections cause an important health and economic burden. Therefore, the need to identify alternative antiviral strategies persists. In this study, we identified non-steroidal estrogens as potent inhibitors of influenza virus due to their interaction with the hemagglutinin protein, preventing viral entry. This activity is maintained in vitro, ex vivo, and in vivo. Therefore, we found a new domain to target on the hemagglutinin and a class of compounds that could be further optimized for influenza treatment.

Antiviral Peptides as Anti-Influenza Agents.

Influenza viruses represent a leading cause of high morbidity and mortality worldwide. Approaches for fighting flu are seasonal vaccines and some antiviral drugs. The development of the seasonal flu vaccine requires a great deal of effort, as careful studies are needed to select the strains to be included in each year's vaccine. Antiviral drugs available against Influenza virus infections have certain limitations due to the increased resistance rate and negative side effects. The highly mutative nature of these viruses leads to the emergence of new antigenic variants, against which the urgent development of new approaches for antiviral therapy is needed. Among these approaches, one of the emerging new fields of "peptide-based therapies" against Influenza viruses is being explored and looks promising. This review describes the recent findings on the antiviral activity, mechanism of action and therapeutic capability of antiviral peptides that bind HA, NA, PB1, and M2 as a means of countering Influenza virus infection.

Influenza Vaccines: Successes and Continuing Challenges.

Influenza vaccines have been available for over 80 years. They have contributed to significant reductions in influenza morbidity and mortality. However, there have been limitations in their effectiveness, in part due to the continuous antigenic evolution of seasonal influenza viruses, but also due to the predominant use of embryonated chicken eggs for their production. The latter furthermore limits their worldwide production timelines and scale. Therefore today, alternative approaches for their design and production are increasingly pursued, with already licensed quadrivalent seasonal influenza vaccines produced in cell cultures, including based on a baculovirus expression system. Next-generation influenza vaccines aim at inducing broader and longer-lasting immune responses to overcome seasonal influenza virus antigenic drift and to timely address the emergence of a new pandemic influenza virus. Tailored approaches target mechanisms to improve vaccine-induced immune responses in individuals with a weakened immune system, in particular older adults.

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

BACKGROUND: 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. METHODS: 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. RESULTS: At least 3 of 7 participants demonstrated confirmed laboratory evidence of sequential infection, with 5 of 7 demonstrating clinical evidence. CONCLUSIONS: 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. CLINICAL TRIALS REGISTRATION: NCT01646138; NCT01971255.

The Hemagglutinin A Stem Antibody MEDI8852 Prevents and Controls Disease and Limits Transmission of Pandemic Influenza Viruses.

Background: MEDI8852 is a novel monoclonal antibody (mAb) that neutralizes both group I and group II influenza A viruses (IAVs) in vitro. We evaluated whether MEDI8852 was effective for prophylaxis and therapy against representative group I (H5N1) and group II (H7N9) pandemic IAVs in mice and ferrets and could be used to block transmission of influenza H1N1pdm09 in ferrets, compared to an irrelevant control mAb R347 and oseltamivir. Methods: MEDI8852 was administered to mice and ferrets by intraperitoneal injection at varying doses, 24 hours prior to intranasal infection with H5N1 and H7N9 viruses for prophylaxis, and 24, 48, and 72 hours post-infection for treatment. A comparison with oseltamivir alone and combination of MEDI8852 and oseltamivir was included in some studies. Survival, weight loss, and viral titers were assessed over a 14-day study period. For the transmission study, naive respiratory contact ferrets received MEDI8852 or R347 prior to exposure to ferrets infected with an H1N1pdm09 virus. Results: MEDI8852 was effective for prophylaxis and treatment of H7N9 and H5N1 infection in mice, with a clear dose-dependent response and treatment with MEDI8852 24, 48, or 72 hours postinfection was superior to oseltamivir for H5N1. MEDI8852 alone was effective treatment for lethal H5N1 infection in ferrets compared to oseltamivir and R347, and MEDI8852 plus oseltamivir was better than oseltamivir alone. MEDI8852 or oseltamivir alone early in infection was equally effective for H7N9 infection in ferrets while the combination yielded similar protection when treatment was delayed. MEDI8852 was able to protect naive ferrets from airborne transmission of H1N1pdm09. Conclusions: MEDI8852, alone or with oseltamivir, shows promise for prophylaxis or therapy of group I and II IAVs with pandemic potential. Additionally, MEDI8852 blocked influenza transmission in ferrets, a unique finding among influenza-specific mAbs.

Limited awareness of animal influenza prevention and control among Dai Lue smallholder farmers in Southwest China.

Awareness of animal influenza and its prevention and control is important for ensuring livestock health, production and welfare. In China, a country stereotyped as a major source of emerging zoonotic infectious diseases, research on the public understanding of animal influenza is limited to the Han, the main ethnic group. The present qualitative study in Southwest China investigated awareness of animal influenza among the Dai, an ethnic minority. The participants (15 men and 10 women, ages 18-83) were smallholder farmers of pigs and poultry in rural areas of Jinghong, Xishuangbanna, Yunnan Province. A mixture of interviews and group discussions took place in homes and villages. The participants were asked about their knowledge of avian influenza (H7N9), swine influenza (H1N1), precautions taken to protect against influenza, procedures when animals were sick and perceived risk of animal influenza. The data were analysed following coding and thematic analysis. The findings demonstrated a limited understanding of animal health and welfare among participants. Specifically, they were largely unaware of animal influenza (H7N9, H1N1) including its causes, symptoms, prevention and treatment. The farmers were also uninformed of the risks they faced and unknowingly engaged in behaviours which increased direct or indirect exposure to infected animals, a risk factor for human infection. They also reported poor usage of veterinary services. In order to guarantee the health, welfare and production of their livestock, immediate action is needed to enable Dai smallholder farmers to prevent and respond to animal influenza effectively and timely.

Swine Worker Precautions During Suspected Outbreaks of Influenza in Swine.

To assess the behavior and precautions that swine workers take during suspected influenza outbreaks in swine, six commercial swine farms in the Midwest U.S. region were visited when influenza outbreaks were suspected in herds during the fall/winter of 2012-2013. Use of personal protective equipment (PPE) and type of task performed by swine workers were recorded based on farm representative reports. Between one to two workers were working on the day of each visit and spent approximately 25 minutes performing work-related tasks that placed them in close contact with the swine. The most common tasks reported were walking the aisles (27%), handling pigs (21%), and handling equipment (21%). The most common PPE were boots (100%), heavy rubber gloves (75%), and dedicated nondisposable clothing (74%). Use of N95 respirators was reported at three farms. Hand hygiene practices were common in most of the farms, but reportedly performed for only 20% to 25% of tasks.

Influenza A virus infection in dogs: Epizootiology, evolution and prevention - A review.

Canine influenza virus (CIV) is an enveloped virus belonging to the genus Influenza virus A within the family Orthomyxoviridae. Prior to 2004, only sporadic outbreaks of canine influenza had been observed in dog populations around the world. However, in 2004 an H3N8 influenza virus of equine origin caused severe respiratory disease in racing greyhounds in Florida; subsequently, cases of dogs affected with various subtypes of CIV have been reported in many countries. Here, we performed a structured review of CIV, including its emergence, evolution and epizootiology. Although CIV causes a disease of low mortality, the potential public health threat it poses due to close contact between dogs and humans highlights the necessity of promoting surveillance for this virus.

Traditional and new influenza vaccines.

The challenges in successful vaccination against influenza using conventional approaches lie in their variable efficacy in different age populations, the antigenic variability of the circulating virus, and the production and manufacturing limitations to ensure safe, timely, and adequate supply of vaccine. The conventional influenza vaccine platform is based on stimulating immunity against the major neutralizing antibody target, hemagglutinin (HA), by virus attenuation or inactivation. Improvements to this conventional system have focused primarily on improving production and immunogenicity. Cell culture, reverse genetics, and baculovirus expression technology allow for safe and scalable production, while adjuvants, dose variation, and alternate routes of delivery aim to improve vaccine immunogenicity. Fundamentally different approaches that are currently under development hope to signal new generations of influenza vaccines. Such approaches target nonvariable regions of antigenic proteins, with the idea of stimulating cross-protective antibodies and thus creating a "universal" influenza vaccine. While such approaches have obvious benefits, there are many hurdles yet to clear. Here, we discuss the process and challenges of the current influenza vaccine platform as well as new approaches that are being investigated based on the same antigenic target and newer technologies based on different antigenic targets.

[COMPARATIVE IMMUNOGENICITY STUDIES OF ADJUVANTS FROM VARIOUS SOURCES AND WITH DIFFERENT MECHANISMS OF ACTION FOR INACTIVATED INFLUENZA VACCINES].

AIM: Direct immunogenicity comparison of adjuvants from various sources and with different mechanisms of action for inactivated influenza vaccines. MATERIALS AND METHODS: Groups of mice were immunized intramuscularly twice with an inactivated whole-virion influenza vaccine based on A/California/07/2009 X-179A (H1N1) strain. The following adjuvants were added to the vaccine (10 in total): aluminium hydroxide, oligonucleotide CpG, complete Freund's adjuvant, poly(lactide-coglycolide) microparticles, monophosphoryl lipid A and polyoxidonium, as well as 2 adjuvants based on characterized chitosan substances with different physical/chemical properties and 2 experimental complex formulations (a multi-component adjuvant and an oil-in-water emulsion based on squalene and tocopherol). Immuogenicity was determined by HAI and MN (MDCK) sera antibodies. RESULTS: Different adjuvants increased immunogenicity of the vaccine against the homologous strain in varying patterns. Experimental complex formulations were the most immunogenic (antibody titer increase reached 48 - 96 times compared with unadjuvanted vaccines). Chitosan based adjuvants showed high immunogenicity. Not all the adjuvants significantly increased immunogenicity, and in some cases even an immunogenicity decrease was noted with the addition of certain adjuvants. CONCLUSION: Research and development of chitosan based adjuvants with characterization and standardization issues addressed, as well as complex adjuvants, both multi-component and emulsion based, are the most promising approaches that could lead to next generation vaccines against influenza and other human and animal infectious diseases.