Antigenic Variants Of Influenza Viruses Research Articles

Longitudinal Analysis of Neuraminidase and Hemagglutinin Antibodies to Influenza A Viruses after Immunization with Seasonal Inactivated Influenza Vaccines.

Neuraminidase (NA)-based immunity could reduce the harmful impact of novel antigenic variants of influenza viruses. The detection of neuraminidase-inhibiting (NI) antibodies in parallel with anti-hemagglutinin (HA) antibodies may enhance research on the immunogenicity and duration of antibody responses to influenza vaccines. To assess anti-NA antibodies after vaccination with seasonal inactivated influenza vaccines, we used the enzyme-linked lectin assay, and anti-HA antibodies were detected in the hemagglutination inhibition assay. The dynamics of the anti-NA antibody response differed depending on the virus subtype: antibodies to A/H3N2 virus neuraminidase increased later than antibodies to A/H1N1pdm09 subtype neuraminidase and persisted longer. In contrast to HA antibodies, the fold increase in antibody titers to NA after vaccination poorly depended on the preexisting level. At the same time, NA antibody levels after vaccination directly correlated with titers before vaccination. A difference was found in response to NA antigen between split and subunit-adjuvanted vaccines and in NA functional activity in the vaccine formulations.

Population immunity in residents of the Kaliningrad region to current antigenic variants of influenza viruses over 2018-2021

The article presents the results of population immunity assessment in residents of the Kaliningrad region to current antigenic variants of influenza viruses over the period of 2018 to 2021. To assess spectrum of influenza types circulating in Kaliningrad region, the real-time PCR method was used using AmpliSense reagent kits. The biological material for the study was obtained from oropharyngeal swabs from the persons who applied to polyclinics with inflammatory diseases of upper respiratory tract, or underwent treatment at the out- or inpatient basis in a healthcare facilities of Kaliningrad region. Humoral immunity was assessed by testing blood sera obtained from healthy residents of Kaliningrad region during periodic prophylactic examinations at the city and regional polyclinics. The study of residents covered all age groups. For the period of 2018-2021, more than 14,000 studies have been carried out. Determination of specific serum antibody titers was carried out by staging the hemagglutination suppression reaction using local influenza antigens (LLC PPDP, St. Petersburg). Antibody titers of 1/40 and higher were considered sufficient to reduce the risk of disease by 50%. During the study period, 2165 cases of influenza were confirmed in the region by laboratory tests. Over the study period, the A(H1N1) pdm serotype proved to be the major strain causing influenza, its proportion reached 57.5%. However, along with A(H1N1) pdm, a significant contribution was also made the A(H3N2) strain to the epidemic process, being the etiological cause of influenza infection in 2019 (42.2%), type B influenza strains being actual in 2020 (42.5%). Over the period of 2018-2021, 420 samples of blood sera from vaccinated and non-vaccinated individuals were tested for specific antibodies. The sera were taken 1-2 months after vaccination and between epidemic rises of influenza (April-May). In vaccinated individuals, the antibody titer was protective in 58.3-64.5%. In this group of persons, the titer sufficient to produce immunity was maintained over the spring/summer period. Among those persons who refused vaccination, a protective antibody titer in the pre-epidemic period was noted in 41.2% of the examined, and during the off-season it was detected in 37.4% of the volunteers. Thus, the A(H1N1) pdm strain was the main etiological factor of influenza in Kaliningrad Region in 2018-2021. The protective level of antibodies in vaccinated population over the period preceding the epidemic peak, was observed 30% more often than in unvaccinated individuals. High incidence of influenza B strains noted in 2020 appears to require a change of specific vaccine preparation.

A fuzzy granular sparse learning model for identifying antigenic variants of influenza viruses

Sparse learning has significant applications in statistics, big data, bioinformatics and machine learning. In big data systems, a large amount of redundant, missing and noisy data cause sparsity, and the rapid changes of information result in uncertainty. Since the traditional sparse learning model is difficult to deal with uncertain data, we propose a Fuzzy Granular Sparse Learning (FGSL) model for identifying antigenic variants of influenza viruses. Firstly, a fuzzy set theory is introduced to measure and granulate the influenza viruses. Some fuzzy granules are induced by a single feature fuzzy granulation. Then, a fuzzy granular vector is constructed from these fuzzy granules, and the fuzzy granular regression is presented. Some constraint norms for granules and granular vectors are proposed, which are two granule norms and four granular vector norms. Therefore, the FGSL model is constructed based on granular regression and constraint norms. The FGSL model includes granular ridge and lasso regressions under different constraint norms. Furthermore, we prove the derivative forms of two granular regression functions, guaranteeing the convergence of the FGSL model. The optimization problem of the FGSL model is discussed and two gradient descent algorithms of the FGSL model are designed. Finally, we employ the FGSL model to serologic data and hemagglutinin sequences for learning antigenicity-associated mutations and inferring antigenic variants. The experimental results confirm some advantages of the FGSL model with fast convergence, low RMSE and strong feature selection ability. We successfully identify antigenic variants of influenza viruses by the FGSL model.

RECENT UPDATES ON MOLECULAR DETECTION OF H9N2 AS LOW PATHOGENIC STRAIN OF AVIAN INFLUENZA VIRUS FROM POULTRY FARMS OF LAHORE, PAKISTAN

H9N2 avian influenza outbreaks have caused great economic losses to the poultry industry in recent decades due to a decrease of egg production, high morbidity, and mortality. Due to different antigenic variants, Influenza virus has become problematical because it has the ability to cross the species barrier. As it is highly pathogenic so its diagnosis and vaccines are of high importance. Hemagglutination inhibition (HI) test is mostly used for subtyping and detection of antibody titer against the virus. Furthermore, its continuous mutations in the HA gene transforms AIV subtype H9N2 (a low pathogenic subtype) into high pathogenic virus subtypes like H5N2 and H7N7 that may have pandemic potential. Thus, it is necessary to identify various antigenic variants of Influenza virus, so it is direly needed to study the HA gene, its attachment to host receptors, the release of genetic material and pathogenicity. In the present study, virus samples from poultry were isolated. Both serological and molecular confirmation was done for 100 samples collected from the different area. They were properly labeled and prepared for the process of egg inoculation in embryonated eggs. The virus was grown in amnioallantoic membrane of embryonated eggs and harvested fluid is then proceeded for confirmatory testing. Hemagglutination and Hemagglutination inhibition testing was done. RNA was extracted by the kit method and cDNA was synthesized. Reverse transcriptase (RTPCR) was performed using specific primer sets and then the PCR product was run on agarose gel. The bands obtained were sent for sequencing.

Anticipating time-dependent antigenic variants of influenza A (H3N2) viruses

Frequent variations in influenza vaccines are necessary to match antigenic variants which appear in influenza epidemics. Antigenic variants of influenza viruses result from frequent mutations in amino acid residues located on their hemagglutinin (HA) proteins. Knowledge of specific changes in these amino acids helps to characterize distinct antigenic variants. In this paper, statistical models are developed and used to investigate changes in amino acids which accompany antigenic variants of epidemiological importance. Amino acid sequences of the HA proteins of influenza A (H3N2) strains isolated from 1968 to 2015 were obtained. The sequences were aligned using Clustal Omega and the number of differences in amino acid residues located on annotated positions of antigenic sites of the HA protein between pairs of strains were determined. These were linked in the statistical models and used to assess the relationship between any pair of influenza strains. The results revealed that both antigenic similarity between strains and the amino acid changes are affected by the time of isolation of the strains. Furthermore, the models predicted that rates of changes in amino acids located on the antigenic sites ranged between 5% and 6% per site per year. The findings of the study suggest that time-dependent antigenic variants of influenza A (H3N2) strains may occur as they evolve. The study has the potential to greatly improve influenza surveillance in as much as it supports vaccine designs.

Detection of Antigenic Variants of Subtype H3 Swine Influenza A Viruses from Clinical Samples.

A large population of genetically and antigenically diverse influenza A viruses (IAVs) are circulating among the swine population, playing an important role in influenza ecology. Swine IAVs not only cause outbreaks among swine but also can be transmitted to humans, causing sporadic infections and even pandemic outbreaks. Antigenic characterizations of swine IAVs are key to understanding the natural history of these viruses in swine and to selecting strains for effective vaccines. However, influenza outbreaks generally spread rapidly among swine, and the conventional methods for antigenic characterization require virus propagation, a time-consuming process that can significantly reduce the effectiveness of vaccination programs. We developed and validated a rapid, sensitive, and robust method, the polyclonal serum-based proximity ligation assay (polyPLA), to identify antigenic variants of subtype H3N2 swine IAVs. This method utilizes oligonucleotide-conjugated polyclonal antibodies and quantifies antibody-antigen binding affinities by quantitative reverse transcription-PCR (RT-PCR). Results showed the assay can rapidly detect H3N2 IAVs directly from nasal wash or nasal swab samples collected from laboratory-challenged animals or during influenza surveillance at county fairs. In addition, polyPLA can accurately separate the viruses at two contemporary swine IAV antigenic clusters (H3N2 swine IAV-α and H3N2 swine IAV-ß) with a sensitivity of 84.9% and a specificity of 100.0%. The polyPLA can be routinely used in surveillance programs to detect antigenic variants of influenza viruses and to select vaccine strains for use in controlling and preventing disease in swine.

Influenza Vaccines: Present and Future

Immunization is the most feasible method for preventing influenza. Vaccination against influenza is recommended for everyone 65 years of age and older and for persons less than 65 years of age who are at risk for developing complications of influenza. Immune correlates of protection have been established, and a global network is in place to monitor the appearance and circulation of antigenic variants of influenza viruses, as well as the appearance of novel subtypes of influenza A. Antigenic and genetic analyses of circulating viruses and testing of serum from vaccine recipients guide vaccine composition updates. The efficacy of influenza vaccines depends in part on the closeness of the antigenic match between the vaccine strain and the epidemic strain. Currently licensed influenza vaccines are trivalent, formalin-inactivated, egg-derived vaccines; their efficacy ranges from 70 to 90% in young, healthy populations when there is a close antigenic match between vaccine strains and epidemic strains. Development of intranasally administered alternative vaccines and improvement of the existing vaccine are areas of active research. A trivalent, ca live vaccine is the most promising LAIV candidate. In a field trial, efficacy rates of LAIV in young children were 96% against influenza A (H3N2) and 91% against influenza B. However, few data are available to compare this formulation of the trivalent ca live vaccine with the trivalent, inactivated vaccine. Influenza vaccine recommendations will most likely be revised on licensure of LAIV; each vaccine may offer distinct advantages in specific populations.

Single-amino-acid substitution in an antigenic site of influenza virus hemagglutinin can alter the specificity of binding to cell membrane-associated gangliosides

Antigenic variants of influenza virus A/Mem/1/71-Bel/42 (H3N1) selected with monoclonal antibodies and having single substitutions in their hemagglutinins were examined for their ability to hemagglutinate and hemolyse erythrocytes coated with different gangliosides. The majority of variants, including one with a substitution near the receptor-binding site (Asn-133----Lys), did not differ from the parent in specificity for receptor molecules. However, a substitution in HA1 at residue 205 (Ser----Tyr), which is distant from the receptor-binding site in antigenic site D, affected hemagglutination and hemolysis of erythrocytes coated with sialyl-paraglobosides. The variant preferentially recognized N-acetylneuraminic acid-alpha 2,6-galactose linkages to sialylparaglobosides, whereas the parent and other variants preferentially recognized N-acetylneuraminic acid-alpha 2,3-galactose linkages. In the trimeric hemagglutinin molecule, residue 205 is located across the subunit interface from the receptor-binding site. The bulky hydrophobic tyrosine in the variant may cause a conformational change in the receptor-binding pocket on the neighboring subunit and influence receptor binding.

Receptor-binding characteristics of monoclonal antibody-selected antigenic variants of influenza virus.

Erythrocytes modified to different extents with periodate were used in hemagglutination assays to investigate the binding properties of antihemagglutinin monoclonal antibody-selected antigenic variants of X-31 influenza virus. The results allowed differentiation of groups of variants and are discussed in relation to the nature of the amino acid substitutions in the variant hemagglutinins and their molecular locations relative to the receptor-binding site.

An 18-amino acid deletion in an influenza neuraminidase

The genes coding for the neuraminidase (NA) enzyme in antigenic variants of influenza virus X-7(F1) (which contains the NA of A/RI/5 +/57) are shorter than other N2 neuraminidase genes. When the parent X-7(F1) virus was cloned at limiting dilution prior to selection of variants with monoclonal antibodies, a minor component with a shorter neuraminidase gene was cloned out, and this deletion was retained in all the variants. Sequence analysis has shown that there is a single deletion, 54 nucleotides long, which occurs in the coding region of the NA, The 18 deleted amino acids are from the stalk region of the protein, which is a thin structure containing the four polypeptide chains of the tetrameric enzyme which separates the enzymatically and antigenically active “head” from the hydrophobic sequence embedded in the viral membrane. The deletion can be seen as a shortened stalk when rosettes of detergent-released NA are examined by electron microscopy, which confirms that the NA stalk is a highly extended structure. Although the more abundant hemagglutinin (HA) would probably extend beyond the shortened NA molecule on the surface of the virus, the NA head is still accessible to all of the available monoclonal antibodies to the different antigenic sites on the molecule. Enzymatic activity toward a small substrate molecule, N-acetyl neuraminyl lactose, is the same for both the parental X-7(F1) virus and the strain having the deleted NA; however, the latter virus is slower to cleave a large substrate, fetuin, and elutes from red cells at a reduced rate.

Evidence for host-cell selection of influenza virus antigenic variants.

Extensive antigenic variability and a capricious epidemiology are characteristics of influenza A and B viruses of man. The haemagglutinin (HA) undergoes frequent and progressive antigenic drift as a result of selection, under immunological pressure, of viruses possessing alterations in the amino acid sequences at specific sites in the molecule. Here we present evidence for an additional selection mechanism for antigenic variants of influenza virus that depends on differing host cell tropisms of virus subpopulations. These studies were initiated after earlier observations of the occurrence of a marked degree of antigenic variation during passage of laboratory strains of influenza virus in eggs and cell cultures (J.C.J., in preparation). We have now shown that cultivation of influenza B viruses in eggs selects subpopulations which are antigenically distinct from virus from the same source grown in mammalian cell cultures. As antigenic characterization of influenza virus strains for epidemiological purposes and for the preparation of influenza vaccines conventionally relies on the cultivation of virus in eggs, our findings may have important practical implications for vaccine design and efficacy.

Determination of the number of nonoverlapping antigenic areas on Hong Kong (H3N2) influenza virus hemagglutinin with monoclonal antibodies and the selection of variants with potential epidemiological significance

Monoclonal antibodies provided evidence for at least three nonoverlapping antigenic areas on the hemagglutinin molecule of A/Mem/V71 (H3N2) influenza virus. This was established by determining the reactivity patterns of 30 different monoclonal antibodies in hemagglutination-inhibition tests and by the failure to select antigenic variants of influenza virus when monoclonal antibodies from two nonoverlapping areas were used in combination. Antigenic analysis showed that most of the variants selected with monoclonal antibodies could not be distinguished from the parental virus with heterogeneous sera, suggesting that they are probably epidemiologically irrelevant. One variant, however, could be distinguished from the parental virus with heterogeneous sera and this variant had a change in sequence at residue 144 of the HAl polypeptide, from glycine in the parent to aspartic acid in the variant. A similar amino acid change has been found in naturally occurring variants at this residue. These studies suggest that some amino acid substitutions are more important than others for producing viruses with epidemiological potential. Antigenic analysis of naturally occurring H3N2 strains with monoclonal antibodies showed that antigenic variation occurs in each nonoverlapping antigenic area of the HA molecule and established that two distinct variants cocirculated in 1968, Hong Kong/1/68 being distinguishable from Aichi/2/68 in at least two antigenic areas. It appears that there may have been two separate lineages of H3N2 viruses, Hong Kong/1/68 giving rise to variants in England and Aichi/2/68 to variants in the USA and Australia.

Antigenic variants of influenza viruses: Marked differences in the frequencies of variants selected with different monoclonal antibodies

Monoclonal antibodies against the hemagglutinin of two influenza A viruses and one influenza B virus were used to analyze the frequencies of antigenic variant subpopulations in cloned virus seeds. Antigenic variants selected with monoclonal antibodies specific for different antigenic determinants were obtained over a wide range of frequencies. With one monoclonal antibody directed to the hemagglutinin of A/PR/8/34 virus antigenic variants were isolated at a frequency of 10- 4.4, whereas another monoclonal antibody, specific for the hemagglutinin of X-31 virus, completely neutralized all infectious virus in the X-31 virus seeds examined, indicating a frequency of antigenic variation, for one seed at least, of less than 10 −8.1.

Induction and characterization of delayed‐type hypersensitivity to influenza virus in mice

AbstractMice infected with an aerosol of influenza virus or immunized with purified UV‐ inactivated whole virus or viral subunits developed delayed‐type hypersensitivity (DTH) to influenza virus. The level of DTH was greatly enhanced when mice were injected intraperitoneally 2 days prior to immunization with 200 mg/kg of cyclophosphamide. The reaction peaked at 24 h after elicitation, had the classical DTH histology, and was transferable by immune cells but not by immune serum. DTH induced in cyclophosphamide‐pretreated mice persisted for at least 40 days after sensitization. DTH induced by deoxycholate‐treated subviral particles (DC particles) or the matrix protein of the influenza virus was type‐specific, i.e. DTH induced by DC particles or matrix protein of type A virus cross‐reacted only with type A virus and not with type B virus. DTH induced by purified hemagglutinin (HA) subunits, on the other hand, showed subtype specificty. Thus, DTH induced by HA of X 31 virus (H3) did not cross‐react with PR 8 virus (H 0). However, DTH induced by HA of one subtype cross‐reacted significantly with the variants of the same subtype. Thus, it appears that the effector T cells of DTH do not discriminate between the antigenic variants of influenza virus that are distinguishable by serology. DTH induced by purified, UV‐ inactivated whole virus showed extensive cross‐reactivity. This nonspecific reaction was shown to be due to host‐derived material, the lipid bilayer. ‐ The theoretical and practical implications of these findings are discussed.

Predetermination by infection and by vaccination of antibody response to influenza virus vaccines.

The antibody orienting effects of prior infections with antigenic variants of influenza viruses were confirmed by studies with monovalent adjuvant vaccines and with polyvalent aqueous and adjuvant preparations. In either case, the predominant antibody response was of a "booster" type, directed against the major antigens of strains of original infection. It was shown that vaccination with appropriate strains, selected as antigenic prototypes, could orient or predetermine subsequent antibody response upon revaccination. Moreover, the effects of exposure by vaccination were found to be durable and to constitute a foundation upon which future antibody dividends could be accumulated. As a result, it seems feasible to induce by vaccination a more lasting broad composite antibody protection against influenza if appropriate preparations and schedules are used.

A serologic recapitulation of past experiences with influenza A; antibody response to monovalent vaccine.

The results of the present study provide a striking demonstration of antibody orientation produced by the dominant antigens of the strains of influenza virus encountered at childhood. The homologous and heterologous antibody response to monovalent vaccines containing swine, PR8, FM1, or Cuppett viruses show that the antibody-forming mechanisms of children born after 1943 are oriented to strains of influenza A-prime; of recruits to strains of influenza A; and of persons over 30 years of age to a strain of swine influenza. It was shown that antibody response to heterotypic viruses appears to provide a serologic index of the amount of experience each of the segments of the general population studied have had with antigenic variants of influenza virus. Finally it was demonstrated that the antibody response of humans to strains of influenza A or influenza A-prime is remarkably uniform for isolates of each antigenic type.

The current status of the control of influenza.

Excerpt It has been my privilege on several occasions in the past 20 years to review before The American College of Physicians the status of knowledge of influenza. The studies of influenza, a dise...

Epidemiologic and immunologic significance of age distribution of antibody to antigenic variants of influenza virus.

The effects on the antibody content of the population which result from repeated exposure to antigenic variants of influenza viruses have been studied by measuring, with many strains, the antibody content of lots of gamma globulin prepared in different years and the patterns of antibody found in sera collected in 1 year from various age groups. In all samples of gamma globulin collected from 1943 through 1951, high levels of antibody were found with strains of Type A and Type B influenza viruses isolated prior to 1941. The highest levels were found in the more recent collections of gamma globulin. Antibodies to A-prime, and to B strains of 1945 and 1952, were present at low levels in gamma globulin collected prior to the isolation of these viruses. A moderate increase in antibody was observed in the gamma globulin of recent years. The pattern of distribution of antibody by age found with most A-prime strains in serum pools exhibited high levels in infancy and childhood, but after the age of 20, little or no antibody was detected. With Type A strains antibody was usually not observed until the 11th year of age. Thereafter, high levels were present until age 20, when the amount of antibody declines to a moderate and relatively constant level which persists throughout life. Antibody against swine influenza virus did not become detectable until the 29th year. The intermediate antigenic character of a few A-prime isolates was reflected in the antibody pattern obtained with them. Antibody was not found until age 13 with the Lee (1940) strain of Type B influenza virus, but thereafter the level was high. With the type B isolates of 1945 and 1952, antibody became measurable at earlier ages. The present data clearly demonstrate that in the early years of life the range of the antibody spectrum is narrow, and that it becomes progressively broader in later life. A striking correlation was found between what is known of the periods of prevalence of certain strains of influenza viruses and the age of the people in whom strain-specific antibodies are currently found. It has been observed that the age at which antibodies to certain strains are first detectable has progressively advanced with the passage of time. From these data the following immunologic thesis is formulated. The antibody which is acquired during the initial infections of childhood is of limited scope and reflects the dominant antigens of the prevailing strains. The immunity conferred by the initial experiences with influenza is also limited. Successive experiences later in life with viruses of related but differing antigenic make-up result in a composite of antibody which is oriented toward a larger number of the common antigens which comprise influenza virus. These experiences confer a broader immunity which limits infection with, and antibody response to, the more recently encountered strains. The antibody-forming mechanisms appear to be oriented by the initial infections of childhood so that exposures later in life to antigenically related strains result in a progressive reinforcement of the primary antibody. The highest cumulative antibody levels detectable in a particular age group tend, therefore, to reflect the dominant antigens of the virus responsible for the childhood infections of that group. Hence the pattern of antibody distribution determined currently in different age groups provides a serologic recapitulation of past infection with antigenic variants of influenza viruses.