Subtype H3N2 Influenza A Viruses: An Unmet Challenge in the Western Pacific

Subtype H3N2 influenza A viruses (A(H3N2)) have been the dominant strain in some countries in the Western Pacific region since the 2009 influenza A(H1N1) pandemic. Vaccination is the most effective way to prevent influenza; however, low vaccine effectiveness has been reported in some influenza seasons, especially for A(H3N2). Antigenic mismatch introduced by egg-adaptation during vaccine production between the vaccine and circulating viral stains is one of the reasons for low vaccine effectiveness. Here we review the extent of this phenomenon, the underlying molecular mechanisms and discuss recent strategies to ameliorate this, including new vaccine platforms that may provide better protection and should be considered to reduce the impact of A(H3N2) in the Western Pacific region.

Evaluation of cytokine production by J774.G8 macrophages after treatment with biotherapics

Introduction: Strains of macrophages, such as murine J774.G8 macrophages, are susceptible to influenza A infection [1]. One of the responses to viral infection involves the production of various types of immunostimulatory cytokines by infected cells [2]. Methods: In the present study, the macrophage strain J774.G8, maintained in RPMI medium, was submitted to treatment with 10% V/V of two different biotherapics prepared from influenza H3N2, both at 30x. Additionally, two control groups were analyzed: macrophages stimulated with water 30x and macrophages without any treatment. Biotherapics were prepared from intact H3N2 influenza virus and H3N2 inactivated by alcohol 70%. The compounding of both biotherapics followed this procedure: one part of viral particles was diluted in 9 parts of sterile distilled water. The 1:10 sample was submitted to 100 mechanical succussions using Autic® Brazilian machine, originating the first dilution, named decimal (1x). 1 ml of this solution was diluted in 9 ml of solvent and was submitted to 100 succussions, generating biotherapic 2x. This procedure was successively repeated, according to Brazilian Homeopathic Pharmacopoeia, to obtain the biotherapic 30x. By the same technique, water vehicle was prepared in the potency of 30x to be used as control. All samples were prepared under sterile and aseptic conditions, using laminar flow cabinet, class II, and were stored in the refrigerator (8ºC), to avoid microbiological contamination. J774.G8 macrophages were stimulated for 2 days, in a total of six stimuli. Immediately before infection with 25 µl of H3N2 influenza virus, the supernatants were collected and frozen at -20 ºC for later analysis. Next, 24 hours after the virus infection, the supernatants were aliquoted and frozen under the same conditions. Three independent experiments were done in triplicate. Analysis of supernatants was performed by flow cytometry using the Mouse Inflammation Kit. The cytokines detected in this experiment were IL-10, IL 12, TNF-α and MCP1. Results: In all cases, there were no significant differences compared to control groups. However, the production of TNF-α detected in macrophages treated by intact and inactivated biotherapics presented a tendency to increase after infection. In fact, similar results were previously detected in other experiments conducted only with the intact biotherapic [3]. The release of the cytokine MCP1 in all experimental situations presented a tendency to decrease after the viral infection when compared to untreated macrophages. No statistically significant difference was detected in the production of IL 12 and IL 10. These experiments will be repeated to confirm the data obtained.

H3N2 Influenza Viruses with 12- or 16-Amino Acid Deletions in the Receptor-Binding Region of Their Hemagglutinin Protein

The hemagglutinin (HA) protein of influenza viruses serves as the receptor-binding protein and is the principal target of the host immune system. The antigenic epitopes in the receptor-binding region are known to tolerate mutations, but here, we show that even deletions of 12 or 16 amino acids in this region can be accommodated.

Impact of amino acid substitution T203I in hemagglutinin on growth characteristics in vitro and hemagglutinin thermostability of A/H3N2 influenza viruses

Background: Russian live attenuated influenza vaccines are a three-component preparations that contain vaccine strains based on current epidemic influenza A/H1N1, A/H3N2 and B strains. Recent influenza viruses A/H3N2 are most susceptible to drift antigenic changes, and therefore, this component of the live attenuated influenza vaccines must be constantly updated. Current vaccine strains of live attenuated influenza vaccines are obtained by the method of classical reassortment using selective factors in developing chicken embryos. During the process of preparation of live attenuated influenza vaccines strains, single reassortants can acquire various egg-adaptive amino acid substitutions in hemagglutinin and neuraminidase the genes responsible for the antigenic correspondence of the vaccine strain to the epidemic parent. These amino acid substitutions can affect the biological properties of the vaccine strain, thereby reducing the effectiveness of this component of live attenuated influenza vaccines. AIM: The aim of the study was to explore the effect of amino acid substitution T203I in hemagglutinin of A/H3N2 influenza viruses on growth characteristics and hemagglutinin thermostability. MATERIALS AND METHODS: For the study, three pairs of A/H3N2 vaccine reassortants were prepared. Reassortants differed from each other by amino acid Thr or Ile at position 203 in the hemagglutinin. The growth properties of vaccine strains were assessed by titration in eggs at 2640C and in a MDCK cell culture at 33C. The thermostability of the hemagglutinin of studied influenza viruses was assessed by determining their ability to agglutinate 1% erythrocytes after exposure to elevated temperatures in the range of 3770C. RESULTS: The amino acid substitution T203I in hemagglutinin in reassortants obtained on the basis of current influenza A/H3N2 viruses acquired during the preparation of vaccine strains does not affect the temperature sensitivity of viruses. It was shown that viruses with an egg-adaptive substitution T203I in hemagglutinin have more pronounced cold-adapted phenotype and a higher reproductive activity in MDCK cell culture, compared to strains without this mutation. It was found that hemagglutinin of reassortants with 203 Ile is more thermostable than with 203 Thr. CONCLUSIONS: Our data indicate that the amino acid substitution of T203I in hemagglutinin in current influenza A/H3N2 viruses does not have a negative effect on biological properties, but improves growth characteristics in eggs and MDCK cells, as well as the thermostability of viruses.

Safety and Immunogenicity of M2-Deficient, Single Replication, Live Influenza Vaccine (M2SR) in Adults

M2SR (M2-deficient single replication) is an investigational live intranasal vaccine that protects against multiple influenza A subtypes in influenza-naïve and previously infected ferrets. We conducted a phase 1, first-in-human, randomized, dose-escalation, placebo-controlled study of M2SR safety and immunogenicity. Adult subjects received a single intranasal administration with either placebo or one of three M2SR dose levels (106, 107 or 108 tissue culture infectious dose (TCID50)) expressing hemagglutinin and neuraminidase from A/Brisbane/10/2007 (H3N2) (24 subjects per group). Subjects were evaluated for virus replication, local and systemic reactions, adverse events (AE), and immune responses post-vaccination. Infectious virus was not detected in nasal swabs from vaccinated subjects. At least one AE (most commonly mild nasal rhinorrhea/congestion) was reported among 29%, 58%, and 83% of M2SR subjects administered a low, medium or high dose, respectively, and among 46% of placebo subjects. No subject had fever or a severe reaction to the vaccine. Influenza-specific serum and mucosal antibody responses and B- and T-cell responses were significantly more frequent among vaccinated subjects vs. placebo recipients. The M2SR vaccine was safe and well tolerated and generated dose-dependent durable serum antibody responses against diverse H3N2 influenza strains. M2SR demonstrated a multi-faceted immune response in seronegative and seropositive subjects.

Antigenic distance between North American swine and human seasonal H3N2 influenza A viruses as an indication of zoonotic risk to humans

Human-to-swine transmission of influenza A virus (IAV) repeatedly occurs, leading to sustained transmission and increased diversity in swine; human seasonal H3N2 introductions occurred in the 1990s and 2010s and were maintained in North American swine. Swine H3N2 were subsequently associated with zoonotic infections, highlighting the need to understand the risk of endemic swine IAV to humans. We quantified antigenic distances between swine H3N2 and human seasonal vaccine strains from 1973 to 2014 using a panel of monovalent antisera raised in pigs in hemagglutination inhibition (HI) assays. Swine H3N2 lineages retained closest antigenic similarity to human vaccine strains from the decade of incursion. Swine lineages from the 1990s were antigenically more similar to human vaccine strains of the mid-1990s but had substantial distance from recent human vaccine strains. In contrast, lineages from the 2010s were closer to human vaccine strains from 2011 and 2014 and most antigenically distant from human vaccine strains prior to 2007. HI assays using ferret antisera demonstrated that swine lineages from the 1990s and 2010s had significant fold-reduction compared with the homologous HI titer of the nearest pandemic preparedness candidate vaccine virus (CVV) or seasonal vaccine strain. The assessment of post-infection and post-vaccination human sera cohorts demonstrated limited cross-reactivity to swine H3N2 from the 1990s, especially in older adults born before 1970s. We identified swine strains to which humans are likely to lack population immunity or are not protected against by a current human seasonal vaccine or CVV to use in prioritizing future human CVV strain selection. IMPORTANCE Human H3N2 influenza A viruses spread to pigs in North America in the 1990s and more recently in the 2010s. These cross-species events led to sustained circulation and increased H3N2 diversity in pig populations. Evolution of H3N2 in swine led to a reduced similarity with human seasonal H3N2 and the vaccine strains used to protect human populations. We quantified the antigenic phenotypes and found that North American swine H3N2 lineages retained more antigenic similarity to historical human vaccine strains from the decade of incursion but had substantial difference compared with recent human vaccine strains. Additionally, pandemic preparedness vaccine strains demonstrated a loss in similarity with contemporary swine strains. Lastly, human sera revealed that although these adults had antibodies against human H3N2 strains, many had limited immunity to swine H3N2, especially older adults born before 1970. Antigenic assessment of swine H3N2 provides critical information for pandemic preparedness and candidate vaccine development.

Competitive evolution of H1N1 and H3N2 influenza viruses in the United States

Seasonal influenza causes vast public health and economic impact globally. The prevention and control of the annual epidemics remain a challenge due to the antigenic evolution of the viruses. Here, we presented a novel modeling framework based on changes in amino acid sequences and relevant epidemiological data to retrospectively investigate the competitive evolution and transmission of H1N1 and H3N2 influenza viruses in the United States during October 2002 and April 2019. To do so, we estimated the time-varying disease transmission rate from the reported influenza cases and the time-varying evolutionary rate of the viruses from the changes in amino acid sequences. By incorporating these time-varying rates into the transmission models, we found that the models could accurately capture the evolutionary transmission dynamics of influenza viruses in the United States. Our modeling results also showed that models incorporating evolutionary change of the virus could provide better modeling performance suggesting the critical role of the evolutionary change of virus on the disease transmission.

A Cross-Reactive Monoclonal Antibody Against Neuraminidases of Both H9N2 and H3N2 Influenza Viruses Shows Protection in Mice Challenging Models

Neuraminidases (NAs) of H9N2 avian influenza virus (AIV) and H3N2 human seasonal influenza virus (HSIV) share similar antigenic structures. However, there are few reports on epitopes shared by these two NAs. We previously reported a monoclonal antibody (mAb) 1G8 against the NA of H9N2 AIV with neuraminidase inhibition (NI) ability. In this study, 1G8 was shown to cross-react with and inhibit the NA of H3N2 HSIV. In a passive transfer experiment, 1G8 provided protection to mice challenged with rescued H1N2 viruses carrying H9N2 NA or H3N2 NA. Mutation at amino acid position 199 was also selected and proved to be crucial for H3N2 HSIV to escape from mAb 1G8. Moreover, we found that residue 199 contributed to inducing broad protective antibodies without the influence of the N-linked glycosylation at amino acid position 200 in NAs. Residues as residue 199, which are not shielded by glycosylation modification, would form ideal epitopes for developing universal vaccine and protective antibodies.

Association between hemagglutination inhibition antibody titers and protection against RT-PCR confirmed influenza illness in children 6−35 months of age: statistical evaluation of a correlate of protection

Background Data from a randomized, controlled efficacy trial of an inactivated quadrivalent influenza vaccine in children 6−35 months of age were used to determine whether hemagglutination inhibition (HI) antibody titer against A/H1N1 and A/H3N2 is a statistical correlate of protection (CoP) for the risk of RT-PCR-confirmed influenza associated with the corresponding strain. Methods The Prentice criteria were used to statistically validate strain-specific HI antibody titer as a CoP. The probability of protection was identified using Dunning's model corresponding to a pre-specified probability of protection at an individual level. The group level protective threshold was identified using Siber's approach, leading to unbiased predicted vaccine efficacy (VE). A case-cohort sub-sample was used for this exploratory analysis. Results Prentice criteria confirmed that HI titer is a statistical CoP for RT-PCR-confirmed influenza. Dunning's model predicted a probability of protection of 49.7% against A/H1N1 influenza and 54.7% against A/H3N2 influenza at an HI antibody titer of 1:40 for the corresponding strain. Higher titers of 1:320 were associated with more than 80% probability of protection. Siber's method predicted VE of 61.0% at a threshold of 1:80 for A/H1N1 and 46.6% at 1:113 for A/H3N2. Conclusions The study validated HI antibody titer as a statistical CoP, by demonstrating that HI titer is correlated with clinical protection against RT-PCR-confirmed influenza associated with the corresponding influenza strain and is predictive of VE in children 6−35 months of age.