Live Influenza Vaccine Research Articles

PECULARITIES OF DEVELOPMENT OF REASSORTANT STRAINS FOR LIVE INFLUENZA VACCINE BASED ON VIRUSES WITH PANDEMIC POTENTIAL

Objective of the research: to characterize the effectiveness of obtaining reassortant strains for live influenza vaccine based on pandemic and potentially pandemic influenza viruses which are new for humans. Materials and methods of the research: influenza virus A(H1N1)pdm09, potentially pandemic A(H3N2)v and A(H5N1) influenza viruses, a master donor virus for the Russian live influenza vaccine. Virological and molecular genetics methods of research. Results: strains with a 6:2 genome composition for live influenza reassortant vaccine based on the pandemic '(H1N1)pdm09 virus and swine influenza A(H3N2)v virus have been successfully obtained. The reassortment of highly pathogenic avian influenza (HPAI) viruses A(H5N1) and human A(H2N2) master donor virus for live attenuated influenza vaccine is complicated by the features of the constellation of their genes. H5N2 reassortants which inherited only HA gene from HPAI viruses of avian influenza were obtained. Despite the impossibility of development of 6:2 reassortants, the phenotypic, preclinical characteristics of the reassortants with the 7:1 genome composition, and their further clinical studies on volunteers have shown that such vaccine strains can be successfully used to prevent diseases caused by avian influenza viruses. Conclusion. When vaccine strains against potentially pandemic influenza viruses are obtained by the classical reassortment technique then the success depends on the mutual constellation of genes of phylogenetically distant far apart parental viruses. In some cases the inheritance by the reassortant of a single HA gene from the antigenic actual virus can be a compromise for LAIV development.

Influenza vaccines: 'tailor-made' or 'one fits all'.

Currently used inactivated influenza vaccines aim at the induction of virus-neutralizing antibodies directed to the variable head domain of the viral hemagglutinin. Although these vaccines are effective against antigenically matching virus strains, they offer little protection against antigenically distinct drift variants or potentially pandemic viruses of alternative subtypes. In the last decades, the threat of novel influenza pandemics has sparked research efforts to develop vaccines that induce more broadly protective immunity. Here, we discuss the immune responses induced by conventional 'tailor-made' inactivated and live influenza vaccines and novel 'one fits all' candidate vaccines able to induce cross-reactive virus-specific antibody and T cell responses and to afford protection to a wider range of influenza viruses.

Express Screening of Reassortants of Influenza Virus B—Candidates for Vaccinating Strains of Live Influenza Vaccine by Using Pyrosequencing

For the rapid preparation of strains of live influenza vaccine, it is important to have a rapid method for screening vaccine reassortants. In this paper, a method of pyrosequencing was used to analyze the genome of reassortants obtained by crossing the donor of attenuation B/Leningrad/14/17/55 and the wild-type virus B/Indiana/25/2015. It has been shown that pyrosequencing allows the composition of the reassortant genome to be evaluated quickly, economically, and with high accuracy.

Modeling of 3D Structure of Chimeric Constructs Based on Hemagglutinin of Influenza Virus and Immunogenic Epitopes of Streptococcus Agalactiae.

A project of an experimental recombinant vector vaccine for prevention of diseases caused by pathogenic streptococci based on ScaAB lipoprotein of Streptococcus agalactiae and a coldadapted strain of live influenza vaccine as a vector was developed. The sequence of ScaAB lipoprotein was analyzed and fragments forming immunodominant epitopes were determined. Chimeric molecules of influenza virus hemagglutinin H7 carrying insertions of bacterial origin were constructed. Based on the results of simulation, the most promising variants were selected; they represented fragments of lipoprotein ScaAB lacking N-terminal domain bound to hemagglutinin via a flexible linker. These insertions should minimally modulate the properties of the influenza strain, while retaining potential immunogenicity to a wide group of pathogenic streptococci.

The Advisory Committee on Immunization Practices recommendation regarding the use of live influenza vaccine: A rejoinder

The Advisory Committee on Immunization Practices recommendation regarding the use of live influenza vaccine: A rejoinder

Influence of Individual Mutations in Genes Coding Internal Proteins of the Influenza A Virus on Formation of Humoral and Cellular Immune Response in Mice

This study addresses a current problem of vaccinal prevention—the development of approaches to increase the immunogenicity of influenza vaccines—and it is directed at studying the effect of mutations responsible for the degree of attenuation of influenza viruses on the formation of an immune response. We conducted the analysis of the humoral and cellular immune response in mice with the introduction of strains of the influenza A virus containing single and combined point mutations alike typical for the attenuation donor A/Leningrad/134/17/57 (H2N2), which is used at present for the preparation of domestic live influenza vaccine. In the study, 13 mutant strains were compared to the attenuation donor (containing all these mutations) and its “wild” predecessor (without mutations). It has been shown that the presence in internal genes of the “wild” virus of single mutations that are typical for the attenuation donor, as well as their combinations, can affect not only the quantitative indices of the humoral immune response, but also the rate of accumulation of virus-specific serum and secretory antibodies. A group of viruses with mutations in the M1 gene was isolated based on the ability to stimulate T cell response. A promising approach to development of ways to increase the immunogenic properties of live influenza vaccines will be a further search for a balanced combination of mutations in M1 and NS2 genes that enhance the stimulation of most of the immuneresponse factors studied, with mutations in genes of polymerase complex that impart attenuating properties to strains.

Creation of live influenza vaccines with the use of site-specific mutagenesis

The introduction of genetic engineering methods into the production of live influenza vaccines provides significant optimization of separate stages of this process. In recent years, special attention has been paid to a method based on the direct incorporation of well-known and well-studied mutations into the genome of epidemic variants of influenza virus. Such virus obtains the ts- and att phenotypes and can be used for the production of live influenza vaccines. The article discusses the applications of this approach and problems associated with its use in medical and veterinary vaccinology.

M2SR, a novel live influenza vaccine, protects mice and ferrets against highly pathogenic avian influenza

The emergence of highly pathogenic avian influenza H5N1 viruses has heightened global concern about the threat posed by pandemic influenza. To address the need for a highly effective universal influenza vaccine, we developed a novel M2-deficient single replication (M2SR) influenza vaccine virus and previously reported that it provided strong heterosubtypic protection against seasonal influenza viruses in mice. In the current study, we assessed M2SR induced protection against H5N1 influenza in mice and ferrets.Mice were intranasally inoculated with M2SR viruses containing the HA and NA from A/Vietnam/1203/2004 (M2SR H5N1) or A/California/07/2009 (M2SR H1N1). All M2SR vaccinated mice survived lethal challenge with influenza A/Vietnam/1203/2004 (H5N1), whereas 40% of mice vaccinated with recombinant H5 HA and none of the naïve controls survived. M2SR H5N1 provided sterile immunity, whereas low levels of virus were detected in the lungs of some M2SR H1N1 vaccinated mice. In contrast, recombinant H5 HA vaccinated mice and naïve controls showed systemic infection.M2SR H5N1 induced strong serum and mucosal antibody responses (IgG and IgA classes) against H5 HA, with high hemagglutination inhibition (HAI) titers. In contrast, while M2SR H1N1 elicited cross-reactive antibodies recognizing the H5 HA2 stalk region or the neuraminidase, no HAI activity against H5N1 virus was detected after M2SR H1N1 immunization.Both M2SR H5N1 and H1N1 also protected ferrets against lethal challenge with A/Vietnam/1203/2004. A prime–boost regimen provided optimal protection with no virus detected in the respiratory tract or brain after challenge. As in the mouse model, only the M2SR H5N1 vaccine induced HAI antibodies against the challenge virus in ferrets, while the M2SR H1N1 was able to provide protection without the induction of HAI antibodies.In summary, effective protection against highly pathogenic H5N1 influenza virus was provided by both homologous H5N1 M2SR and heterologous H1N1 M2SR demonstrating the cross-protective attributes of the M2SR platform.

Generalized herd effects and vaccine evaluation: impact of live influenza vaccine on off-target bacterial colonisation

Interactions between pathogens and commensal microbes are major contributors to health and disease. Infectious diseases however are most often considered independent, viewed within a one-host one-pathogen paradigm and, by extension, the interventions used to treat and prevent them are measured and evaluated within this same paradigm. Vaccines, especially live vaccines, by stimulating immune responses or directly interacting with other microbes can alter the environment in which they act, with effects that span across pathogen species. Live attenuated infl uenza vaccines for example, while safe, increase upper respiratory tract bacterial carriage density of important human commensal pathogens like Streptococcus pneumoniae and Staphylococcus aureus. Further, by altering the ecological niche and dynamics of phylogenetically distinct microbes within the host, vaccines may unintentionally affect transmission of non-vaccine targeted pathogens. Thus, vaccine effects may span across species and across scales, from the individual to the population level. In keeping with traditional vaccine herd-effects that indirectly protect even unvaccinated individuals by reducing population prevalence of vaccine-targeted pathogens, we call these cross-species cross-scale effects "generalized herd-effects". As opposed to traditional herd-effects, "generalized" relaxes the assumption that the effect occurs at the level of the vaccine-target pathogen and "herd effect" implies, as usual, that the effects indirectly impact the population at large, including unvaccinated bystanders. Unlike traditional herd-effects that decrease population prevalence of the vaccine-target, generalized herd-effects may decrease or increase prevalence and disease by the off-target pathogen. LAIV, for example, by increasing pneumococcal density in the upper respiratory tract of vaccine recipients, especially children, may increase pneumococcal transmission and prevalence, leading to excess pneumococcal invasive disease in the population, especially among the elderly and others most susceptible to pneumococcal disease. However, these effects may also be beneficial, for example the large reductions in all-cause mortality noted following measles vaccines. Here we discuss evidence for these novel vaccine effects and suggest that vaccine monitoring and evaluation programs should consider generalized herd effects to appreciate the full impacts of vaccines, beneficial or detrimental, across species and scales that are inevitably hiding in plain sight, affecting human health and disease.

INCLUSION OF SITE-SPECIFIC MUTATIONS INTO CONSERVATIVE SEGMENTS OF РА-GENE RESULTS IN ATTENUATION OF VIRULENT INFLUENZA VIRUS STRAIN A/WSN/33

Study the possibility of obtaining attenuated variants of influenza virus by including specially selected site-specific mutations into a conservative sequence of PA-gene (terminal segment of COOH-domain of the PA-gene) of a virulent strain. A/ WSN/33 - a virulent strain of influenza virus was used in the study. Inclusion of site-specif- ic mutations into PA-gene of the A/WSN/33 virulent strain was carried out using a two-step mutation PCR. Cloning was carried out using GoldenGate reaction. 8-plasmid transfection system based on pHW2000 vector was used. Transformation was carried out in rubidium competent bacterial cells of DH5(α strain. Transfection was done using Lipofectamine LTX (Invitrogen) reagentin a 293T and MDCK cells' co-culture. Transfectants with F658A substitution in the COOH-domain of the PA-gene were shown to acquire ts-phenotype and sharply reduce the ability to reproduce in mice lungs. Introduction of F658A substitution into COOH-domain of the PA-gene in combination with introduction of ts-mutations from ca influenzavirus strains into the genome ofthe virulent strain resulted in obtaining transfectants that have phenotypic characteristics typical for live influenza vaccine candidates. The ability to obtain attenuated variants of influenza viruses by introducing spe- cially selected site-specific mutations into conservative sequence of the PA-gene is shown.

Direct and indirect effects of influenza vaccination

BackgroundAfter vaccination, vaccinees acquire some protection against infection and/or disease. Vaccination, therefore, reduces the number of infections in the population. Due to this herd protection, not everybody needs to be vaccinated to prevent infections from spreading.MethodsWe quantify direct and indirect effects of influenza vaccination examining the standard Susceptible-Infected-Recovered (SIR) and Susceptible-Infected-Recovered-Susceptible (SIRS) model as well as simulation results of a sophisticated simulation tool which allows for seasonal transmission of four influenza strains in a population with realistic demography and age-dependent contact patterns.ResultsAs shown analytically for the simple SIR and SIRS transmission models, indirect vaccination effects are bigger than direct ones if the effective reproduction number of disease transmission is close to the critical value of 1. Simulation results for 20–60% vaccination with live influenza vaccine of 2–17 year old children in Germany, averaged over 10 years (2017–26), confirm this result: four to seven times as many influenza cases are prevented among non-vaccinated individuals as among vaccinees. For complications like death due to influenza which occur much more frequently in the unvaccinated elderly than in the vaccination target group of children, indirect benefits can surpass direct ones by a factor of 20 or even more than 30.ConclusionsThe true effect of vaccination can be much bigger than what would be expected by only looking at vaccination coverage and vaccine efficacy.

Highly reproductive attenuated H2N2 and H7N9 reassortants on the basis of A/Hong Kong/1/68/162/35 donor virus.

Reassortants with surface antigens from potentially pandemic A/H2N2 and A/H7N9 influenza viruses were created on the basis of attenuated and highly reproductive A/Hong Kong/1/68/162/35(H3N2) donor virus obtained in the Research institute of influenza. High reproductive activity of reassortant viruses and immunogenicity of live and inactivated influenza vaccines based on these viruses indicate the possibility to use obtained reassortants for production of live and inactivated vaccines against potentially pandemic influenza A viruses.

Especial Features of Immune Answer of Mice, Immunized by Different Types of Live Influenza Vaccines Candidate on Infection, Caused by Virulent Influenza Strain

Relevance. Live influenza vaccines are highly effective and currently used in the Russian Federation and the United States. Goal. To investigate especial features of immune response of mice, immunized by different types of live influenza vaccines and infected later by virulent influenza strain. Materials and methods. Mice were immunized by two types of live influenza vaccines candidate: cold-adapted (CA) reassortant, which inherited the 6 «internal» genes from the CA donor A/Krasnodar/101/35/59 (H2N2) and 2 genes encoding the surface proteins HA and NA from the virulent strain A/WSN/33 (H1N1) and site-specific mutants on the basis of A/WSN/33 strain, in the genome of which has been included mutations from genes of CA strains of influenza virus, encoding proteins of the polymerase complex. Immunized mice were then infected by a virulent A/WSN/33 strain of influenza virus. Results. It was shown that CA reassortant RKr35/WSN/33 and site-specific mutants Tr. № 5 and № 8 in contrast to the virulent A/WSN/33 strain were characterized by pronounced ts-phenotype and att-phenotype. Both types of live vaccines after two-time intranasal immunization induced in mice a relatively low level of humoral antibodies (log 2 4,5 ± 1,2 - log 2 6,0 ± 0,7). Despite the low level of induction of humoral response both types of live vaccines had similar marked protective efficiency. However, animals immunized with CA reassortant was characterized by a significant weight loss after infection with a virulent influenza strain (25%), while the infection of animals, immunized with site-specific mutants, by a similar dose of virulent flu strain has very little impact on their weight characteristics (8 - 13%). Conclusion. The data obtained indicate that antibodies to surface proteins of the virion, induced in the process of immunization, slightly inhibited the initial replication of the virulent strain and suggest that these differences in the symptoms of the infection depend on the characteristics of activated T-cell immunity. This circumstance could have a significant impact on metabolic processes in organism of infected animals.

Live Attenuated Flu Vaccine Offers No Extra Community Benefit

Intranasal live attenuated influenza vaccine has been more immunogenic than intramuscular inactivated vaccine among children in clinical trials. This finding has led to the assumption that the live vaccine also should be a more powerful inducer of herd immunity. Canadian researchers studied the community-wide performance of both vaccines in 52 rural Hutterite colonies — tiny religious settlements composed …

Generation and protective efficacy of a cold-adapted attenuated avian H9N2 influenza vaccine.

To prevent H9N2 avian influenza virus infection in chickens, a long-term vaccination program using inactivated vaccines has been implemented in China. However, the protective efficacy of inactivated vaccines against antigenic drift variants is limited, and H9N2 influenza virus continues to circulate in vaccinated chicken flocks in China. Therefore, developing a cross-reactive vaccine to control the impact of H9N2 influenza in the poultry industry remains a high priority. In the present study, we developed a live cold-adapted H9N2 influenza vaccine candidate (SD/01/10-ca) by serial passages in embryonated eggs at successively lower temperatures. A total of 13 amino acid mutations occurred during the cold-adaptation of this H9N2 virus. The candidate was safe in chickens and induced robust hemagglutination-inhibition antibody responses and influenza virus–specific CD4+ and CD8+ T cell immune responses in chickens immunized intranasally. Importantly, the candidate could confer protection of chickens from homologous and heterogenous H9N2 viruses. These results demonstrated that the cold-adapted attenuated H9N2 virus would be selected as a vaccine to control the infection of prevalent H9N2 influenza viruses in chickens.

Strategies to minimize vaccine errors

Mary S. Hayney, PharmD,MPH, BCPS, Professor of Pharmacy, School of Pharmacy, University of Wisconsin, Madison (mary. hayney@wisc.edu) and John D. Grabenstein, PhD, Executive Director, Global Health & Medical Affairs, Merck Vaccines (john_grabenstein@merck.com). Over the past 20 years, the number of vaccine-preventable diseases has nearly doubled and, therefore, the number of vaccine doses has increased for both children and adults.1,2 As immunizations increase, the potential for vaccine errors also increases. Despite robust vaccine adverse event reporting, vaccine errors are often underreported. The Vaccine Adverse Events Reporting System (VAERS) used by the U.S. Food and Drug Administration and the Centers for Disease Control and Prevention (CDC) accepts all reports related to vaccines, including vaccine errors; however, the reporting form is not designed to capture data elements to help describe and analyze vaccine errors.3 To increase and standardize event reporting, the Institute for Safe Medication Practices (ISMP) created the national Vaccine Errors Reporting Program (VERP). With its decades of experience, ISMP can analyze vaccine errors and provide recommendations to increase safety and decrease vaccine errors.4 From 2000 to 2013, 20,585 errors were reported to VAERS. Twenty-five percent of those vaccine errors also reported an adverse health event related to the error, of which 8% were serious. The most common causes of vaccine errors were inappropriate schedule, storage, and dispensing, and wrong vaccine (Table 1). Deviations from the dosing schedule were most common with quadrivalent human papillomavirus (HPV4) and pentavalent rotavirus (RV5), where the administration of second and third doses occurred earlier than recommended. Storage and dispensing errors included 2 main categories: administration of expired vaccine and incorrectly stored vaccine. Live influenza vaccine (LAIV) and zoster vaccine were

Enhancement of the safety of live influenza vaccine by attenuating mutations from cold-adapted hemagglutinin

In our previous study, X-31ca-based H5N1 LAIVs, in particular, became more virulent in mice than the X-31ca MDV, possibly by the introduction of the surface antigens of highly pathogenic H5N1 influenza virus, implying that additional attenuation is needed in this cases to increase the safety level of the vaccine. In this report we suggest an approach to further increase the safety of LAIV through additional cold-adapted mutations in the hemagglutinin. The cold-adaptation of X-31 virus resulted in four amino acid mutations in the HA. We generated a panel of 7:1 reassortant viruses each carrying the hemagglutinins with individual single amino acid mutations. We examined their phenotypes and found a major attenuating mutation, N81K. This attenuation marker conferred additional temperature-sensitive and attenuation phenotype to the LAIV. Our data indicate that the cold-adapted mutation in the HA confers additional attenuation to the LAIV strain, without compromising its productivity and immune response.

Genetic analysis of attenuation markers of cold-adapted X-31 influenza live vaccine donor strain

Cold-adapted live attenuated influenza vaccines (CAIVs) have been considered as a safe prophylactic measure to prevent influenza virus infections. The safety of a CAIV depends largely on genetic markers that confer specific attenuation phenotypes. Previous studies with other CAIVs reported that polymerase genes were primarily responsible for the attenuation. Here, we analyzed the genetic mutations and their phenotypic contribution in the X-31 ca strain, a recently developed alternative CAIV donor strain. During the cold-adaptation of its parental X-31 virus, various numbers of sequence changes were accumulated in all six internal genes. Phenotypic analysis with single-gene and multiple-gene reassortant viruses suggests that NP gene makes the largest contribution to the cold-adapted (ca) and temperature-sensitive (ts) characters, while the remaining other internal genes also impart attenuation characters with varying degrees. A balanced contribution of all internal genes to the attenuation suggests that X-31 ca could serve as an ideal master donor strain for CAIVs preventing influenza epidemics and pandemics.

Potentially Pandemic Live Influenza Vaccines Based on Russian Master Donor Virus are Genetically Stable after Replication in Humans

Stability of attenuating mutations is an important premise for live attenuated influenza vaccine (LAIV). The presence of multiple mutations in internal gene segments of live attenuated viruses contributes to the stability of their genomes and consistent phenotypic properties. This study describes the evaluation of results of clinical isolates obtained in Phase I clinical trials of three Russian LAIVs against potentially pandemic influenza viruses which may cause serious and fatal disease in humans. As part of clinical trials, nasal swabs were tested for vaccine virus shedding, for temperature sensitivity and cold–adaptation and for nucleotide sequence. Vaccine viruses isolated from the vaccinated subjects were shown to retain phenotypic characteristics and attenuating mutations. These data suggest genetic stability of vaccine virus after replication in humans. In addition, no vaccine virus was detected in the placebo groups indicating the lack of person–to–person transmission.

Разработка холодоадаптированного реассортантного вируса гриппа А/H6N1 на основе донора аттенуации A/Ленинград/134/17/57 (H2N2) и его генотипирование методом анализа кривых плавления высокого разрешения (HRM-анализ)

Холодоадаптированный реассортантный штамм вируса гриппа А/17/серебристая чайка/Сарма/2006/887 (H6N1) получен на основе донора аттенуации А/Ленинград/134/17/57 (H2N2) методом генетической реассортации в куриных эмбрионах. Структура генома реассортанта была проанализирована методом ПЦР в реальном времени с последующим анализом кривых плавления высокого разрешения (high resolution melting, HRM-анализ) при использовании интеркалирующего флуоресцентного красителя EvaGreen. Анализ кривых плавления ДНК копий РНК фрагментов (PB2, PB1, PA, NP, М, NS) показал, что реассортантный вирус гриппа А/17/серебристая чайка/Сарма/2006/887 (H6N1) унаследовал гены внутренних и неструктурных белков от донора аттенуации. По антигенным свойствам реассортант соответствовал вирусу гриппа птиц А/серебристая чайка/Сарма/51с/2006 (H6N1). Изучение фенотипических свойств реассортанта показало, что вирус А/17/ серебристая чайка/Сарма/2006/887 (H6N1) характеризуется температурочувствительностью и холодоадаптированностью в куриных эмбрионах и аттенуирован для мышей при интраназальном введении. Данный реассортант может быть рекомендован кандидатом в вакцинные штаммы для живой гриппозной вакцины для людей.