Master donor viruses A/Leningrad/134/17/57 (H2N2) and B/USSR/60/69 and derived reassortants used in live attenuated influenza vaccine (LAIV) do not display neurovirulent properties in a mouse model

Demonstration of the absence of neurovirulent properties of reassortant viruses contained in live attenuated influenza vaccine (LAIV) is a regulatory requirement. A mouse model was used to detect neurovirulent properties of the cold-adapted, temperature-sensitive and attenuated influenza master donor viruses (MDVs) A/Leningrad/134/17/57 (H2N2) and B/USSR/60/69 and derived reassortant influenza viruses. A/NWS/33 (H1N1), which is known to be neurovirulent in mice, was used as a positive control. Under conditions where the positive control virus induced symptoms of disease and showed viral replication in the upper respiratory tract as well as in the brain, replication of the influenza master donor viruses and reassortant influenza A and B viruses was limited to the upper respiratory tract where they were administered. None of the mice inoculated with MDVs or reassortant influenza viruses suffered from disease, and no virus or viral replication was observed in the brains of these mice. The results demonstrate the absence of neurovirulent properties of the MDVs and reassortant influenza viruses derived therefrom used in LAIV.     

Natural co-infection of influenza A/H3N2 and A/H1N1pdm09 viruses resulting in a reassortant A/H3N2 virus

BackgroundDespite annual co-circulation of different subtypes of seasonal influenza, co-infections between different viruses are rarely detected. These co-infections can result in the emergence of reassortant progeny.Study designWe document the detection of an influenza co-infection, between influenza A/H3N2 with A/H1N1pdm09 viruses, which occurred in a 3 year old male in Cambodia during April 2014. Both viruses were detected in the patient at relatively high viral loads (as determined by real-time RT-PCR CT values), which is unusual for influenza co-infections. As reassortment can occur between co-infected influenza A strains we isolated plaque purified clonal viral populations from the clinical material of the patient infected with A/H3N2 and A/H1N1pdm09.ResultsComplete genome sequences were completed for 7 clonal viruses to determine if any reassorted viruses were generated during the influenza virus co-infection. Although most of the viral sequences were consistent with wild-type A/H3N2 or A/H1N1pdm09, one reassortant A/H3N2 virus was isolated which contained an A/H1N1pdm09 NS1 gene fragment. The reassortant virus was viable and able to infect cells, as judged by successful passage in MDCK cells, achieving a TCID₅₀ of 10⁴/ml at passage number two. There is no evidence that the reassortant virus was transmitted further. The co-infection occurred during a period when co-circulation of A/H3N2 and A/H1N1pdm09 was detected in Cambodia.ConclusionsIt is unclear how often influenza co-infections occur, but laboratories should consider influenza co-infections during routine surveillance activities.     

The influence of the multi-basic cleavage site of the H5 hemagglutinin on the attenuation, immunogenicity and efficacy of a live attenuated influenza A H5N1 cold-adapted vaccine virus

A recombinant live attenuated influenza virus ΔH5N1 vaccine with a modified hemagglutinin (HA) and intact neuraminidase genes from A/Vietnam/1203/04 (H5N1) and six remaining genome segments from A/Ann Arbor/6/60 (H2N2) cold-adapted (AA ca) virus was previously shown to be attenuated in chickens, mice and ferrets. Evaluation of the recombinant H5N1 viruses in mice indicated that three independent factors contributed to the attenuation of the ΔH5N1 vaccine: the attenuating mutations specified by the AA ca loci had the greatest influence, followed by the deletion of the H5 HA multi-basic cleavage site (MBS), and the constellation effects of the AA genes acting in concert with the H5N1 glycoproteins. Restoring the MBS in the H5 HA of the vaccine virus improved its immunogenicity and efficacy, likely as a consequence of increased virus replication, indicating that removal of the MBS had a deleterious effect on the immunogenicity and efficacy of the ΔH5N1 vaccine in mice.     

The hemagglutinin protein of influenza A/Vietnam/1203/2004 (H5N1) contributes to hyperinduction of proinflammatory cytokines in human epithelial cells

Live attenuated influenza A/Vietnam/1203/04 (H5N1) (VN04 cold adapted [ca]) and A/Hong Kong/213/03 (H5N1) (HK03 ca) vaccine viruses were compared with the A/New Caledonia/20/99 (H1N1) (NC99 ca) seasonal vaccine virus for induction of host gene expression in infected human epithelial cells. Levels of proinflammatory cytokines and interferon-related genes were significantly upregulated in VN04 ca virus-infected A549 cells compared to cells infected with the HK03 ca and NC99 ca viruses as examined by microarray analysis and confirmed by quantitative RT-PCR and ELISA assays. Further mapping studies demonstrated that the hemagglutinin (HA) protein of the VN04 ca virus contributed to the hyperinduction of cytokines. The inactivated viruses could also induce the production of the cytokines and chemokines, albeit at a much lower level than live viruses. Compared to HK03 ca virus, VN04 ca virus differs by 9 amino acids including an additional glycosylation site at residue 158N of the HA protein and a shortened stalk in the neuraminidase (NA) protein. Increased cytokine production by HK03 ca virus was only observed when HK03 ca virus acquired an additional glycosylation in the HA protein and when its NA protein was replaced by that of VN04. Thus, our data indicate that the HA protein and its interaction with the NA protein play a role in triggering cytokine responses. The full implications of cytokine induction in vaccine virus-induced immune responses remain to be explored.     

Evaluation of Influenza A/Hong Kong/123/77 (H1N1) ts-1A2 and Cold-Adapted Recombinant Viruses in Seronegative Adult Volunteers

Two attenuated influenza A donor viruses, the A/Udorn/72 ts-1A2 and the A/Ann Arbor/6/60 cold-adapted (ca) viruses, are being evaluated for their ability to reproducibly attenuate each new variant of influenza A virus to a specific and desired level by the transfer of one or more attenuating genes. Each of these donor viruses has been able to attenuate influenza A viruses belonging to the H3N2 subtype by the transfer of one or more attenuating genes. To determine whether these two donor viruses could attenuate a wild-type virus that belonged to a different influenza A subtype, ts-1A2 and ca recombinants of a wild-type virus representative of the A/USSR/77 (H1N1) Russian influenza strain were prepared and evaluated in adult doubly seronegative volunteers at several doses. The recombinants derived from both donor viruses were attenuated for the doubly seronegative adults. Less than 5% of infected vaccinees developed a febrile or systemic reaction, whereas five of six recipients of wild-type virus developed such a response. The 50% human infectious dose (HID₅₀) for each recombinant was approximately 10^5.0 50% tissue culture infective doses. The virus shed by the ts-1A2 and ca vaccinees retained the ts or ca phenotype, or both. This occurred despite replication of the recombinant viruses for up to 9 days. No evidence for transmission of the ca or ts-1A2 recombinant virus to controls was observed. A serum hemagglutination inhibition response was detected in less than 50% of the infected vaccinees. However, with the more sensitive enzyme-linked immunosorbent assay, a serological response was detected in 100% of the ca vaccinees given 300 HID₅₀ and approximately 70% of ca or ts vaccinees who received 10 to 32 HID₅₀ of virus. These results indicate that the recombinants derived from both donor viruses were satisfactorily attenuated and were stable genetically after replication in doubly seronegative adults although they induced a lower serum hemagglutination inhibition response than that found previously for H3N2 ts and ca recombinants.     

Differences in transmissibility and pathogenicity of reassortants between H9N2 and 2009 pandemic H1N1 influenza A viruses from humans and swine

Both H9N2 subtype avian influenza and 2009 pandemic H1N1 viruses (pH1N1) can infect humans and pigs, which provides the opportunity for virus reassortment, leading to the genesis of new strains with potential pandemic risk. In this study, we generated six reassortant H9 viruses in the background of three pH1N1 strains from different hosts (A/California/04/2009 [CA04], A/Swine/Jiangsu/48/2010 [JS48] and A/Swine/Jiangsu/285/2010 [JS285]) by replacing either the HA (H9N1-pH1N1) or both the HA and NA genes (H9N2-pH1N1) from an h9.4.2.5-lineage H9N2 subtype influenza virus, A/Swine/Taizhou/5/08 (TZ5). The reassortant H9 viruses replicated to higher titers in vitro and in vivo and gained both efficient transmissibility in guinea pigs and increased pathogenicity in mice compared with the parental H9N2 virus. In addition, differences in transmissibility and pathogenicity were observed among these reassortant H9 viruses. The H9N2-pH1N1viruses were transmitted more efficiently than the corresponding H9N1-pH1N1 viruses but showed significantly decreased pathogenicity. One of the reassortant H9 viruses that were generated, H9N-JS48, showed the highest virulence in mice and acquired respiratory droplet transmissibility between guinea pigs. These results indicate that coinfection of swine with H9N2 and pH1N1viruses may pose a threat for humans if reassortment occurs, emphasizing the importance of surveillance of these viruses in their natural hosts.     

Genetic bases of the temperature-sensitive phenotype of a master donor virus used in live attenuated influenza vaccines: A/Leningrad/134/17/57 (H2N2)

Trivalent live attenuated influenza vaccines whose type A components are based on cold-adapted A/Leningrad/134/17/57 (H2N2) (caLen17) master donor virus (MDV) have been successfully used in Russia for decades to control influenza. The vaccine virus comprises hemagglutinin and neuraminidase genes from the circulating viruses and the remaining six genes from the MDV. The latter confer temperature-sensitive (ts) and attenuated (att) phenotypes. The ts phenotype of the vaccine virus is a critical biological determinant of attenuation of virulence. We developed a plasmid-based reverse genetics system for MDV caLen17 to study the genetic basis of its ts phenotype. Mutations in the polymerase proteins PB1 and PB2 played a crucial role in the ts phenotype of MDV caLen17. In addition, we show that caLen17-specific ts mutations could impart the ts phenotype to the divergent PR8 virus, suggesting the feasibility of transferring the ts phenotype to new viruses of interest for vaccine development.     

The generation and characteristics of reassortant influenza A virus with H5 hemagglutinin and other genes from the apathogenic virus H6N2

The experimental reassortant vaccine strain VN-gull (H5N2) containing H5 hemagglutinin (HA) with a removed polybasic site in the connecting peptide and other genes from the apathogenic H6N2 virus A/gull/Moscow/3100/2006 (gull/M) was obtained using a two-step protocol. At Step 1, the reassortant with HA of A/Vietnam/1203/04-PR8/ CDC-RG and other genes from cold-adapted A/Leningrad/17/47 (VN-Len) viruses was generated due to selection with antibody to H2N2 at 26 degrees C. At Step 2, the reassortant VN-gull was obtained by replacing all genes from Len with those from gull/M due to selection with antibody to H6N2 at 39 degrees C. The reassortant VN-Len was apathogenic and the reassortant VN-gull was weakly virulent in mice. Both gave rise to specific antibodies and 4 weeks after single inoculation they provided complete protection against further challenge with highly pathogenic HSN1 virus A/chicken/Kurgan/3/05 (H5N1) (Ku-Len). The chickens infected with live VN-gull virus showed neither clinical symptoms, nor fecal virus excretion; nevertheless, they gave rise to antibodies and were protected from the further challenge with A/chicken/Kurgan/3/2005. The high yield, safety, and protectivity of VN-Len and Ku-Len made them promising strains for the production of inactivated and live vaccines against H5N1 viruses.     

Evaluation of live avian-human reassortant influenza A H3N2 and H1N1 virus vaccines in seronegative adult volunteers.

An avian-human reassortant influenza A virus deriving its genes coding for the hemagglutinin and neuraminidase from the human influenza A/Washington/897/80 (H3N2) virus and its six "internal" genes from the avian influenza A/Mallard/NY/6750/78 (H2N2) virus (i.e., a six-gene reassortant) was previously shown to be safe, infectious, nontransmissible, and immunogenic as a live virus vaccine in adult humans. Two additional six-gene avian-human reassortant influenza viruses derived from the mating of wild-type human influenza A/California/10/78 (H1N1) and A/Korea/1/82 (H3N2) viruses with the avian influenza A/Mallard/NY/78 virus were evaluated in seronegative (hemagglutination inhibition titer, less than or equal to 1:8) adult volunteers for safety, infectivity, and immunogenicity to determine whether human influenza A viruses can be reproducibly attenuated by the transfer of the six internal genes of the avian influenza A/Mallard/NY/78 virus. The 50% human infectious dose was 10(4.9) 50% tissue culture infectious doses for the H1N1 reassortant virus and 10(5.4) 50% tissue culture infectious doses for the H3N2 reassortant virus. Both reassortants were satisfactorily attenuated with only 5% (H1N1) and 2% (H3N2) of infected vaccines receiving less than 400 50% human infectious doses developing illness. Consistent with this level of attenuation, the magnitude of viral shedding after inoculation was reduced 100-fold (H1N1) to 10,000-fold (H3N2) compared with that produced by wild-type virus. The duration of virus shedding by vaccines was one-third that of controls receiving wild-type virus. At 40 to 100 50% human infectious doses, virus-specific immune responses were seen in 77 to 93% of volunteers. When vaccinees who has received 10(7.5) 50% tissue culture infectious doses of the H3N2 vaccine were experimentally challenged with a homologous wild-type human virus only 2 of 19 (11%) vaccinees became ill compared with 7 of 14 (50%) unvaccinated seronegative controls ( P < 0.025; protective efficacy, 79%). Thus, three different virulent human influenza A viruses have been satisfactorily attenuated by the acquisition of the six internal genes of the avian influenza A/Mallard/NY/78 virus. The observation that this donor virus can reproducibly attenuate human influenza A viruses indicates that avian-human influenza A reassortants should be further studied as potential live influenza A virus vaccines.     

Correlation of polymerase replication fidelity with genetic evolution of influenza A/Fujian/411/02(H3N2) viruses

Influenza virus evolves continuously through mutations presumed to result from evolutionary pressure driving viral replication. This study examined the relationship between the genetic evolution and replication fidelity of influenza viruses. Analysis of influenza sequences from National Centre for Biotechnology Information (NCBI) database revealed a gradual decrease in the rate of genetic evolution of A/Fujian/411/02(H3N2)-like variants after the emergence and predominance of the A/H3N2 Fujian strain in 2002. This decrease may be related to an increase in replication fidelity, which was investigated by assessing mutation frequencies of reassortant viruses carrying the PB1 segment of Fujian variants isolated between 2003 and 2005 in a sequencing-based plaque assay. The data revealed a threefold decrease in substitution per site of the reassortant viruses carrying the Fujian PB1 segments isolated in 2004-2005 compared with those circulating in 2003. The decrease in mutation frequency paralleled a decrease in genetic evolution of the Fujian variants from the NCBI database. This correlation implicates changes in the polymerase replication fidelity as contributing to altered genetic evolution of influenza viruses.     

The inoculative properties of cold-adapted reassortant A(H5N2) influenza strain during intranasal administration to mice

Classical genetic reassortant techniques were used to have a cold-adapted (ca) reassortant A/17/Duck/Potsdam/86/92 (H5N2) that inherited the hemagglutinin (HA) gene from the nonpathogenic avian virus A/Duck/Potsdam/ 1402-6186 (H5N2) and the genes of neuraminidase (NA) and non-glycated proteins from the ca attenuation donor A/Leningrad/134/17/57 (H2N2). All experiments were performed under increased biological protection (BSV-3+). The reassortant and parent H5N2 virus were non-pathogenic to Balb/c mice, the reassortant replication in the murine nasal passages (3.5 Ig EID50/ml) being higher than that in the lung (2.1 lg EID50/ml). Intranasal inoculation of mice with reassortant A/17/Duck/Potsdam/86/92 caused an immune response to both homological H5N2 virus and antigenically differing variants of influenza A (H5N1) virus isolated from humans in 1997 and 2003. The mice intranasally immunized with the ca reassortant were protected against fatal infection with the highly pathogenic A/Hong Kong/483/9797 (H5N1) virus and against infection with A/Hong Kong/213/03(H5N1) virus (80 and 100%, respectively).     

Persistence of Q strain of H2N2 influenza virus in avian species: Antigenic,biological and genetic analysis of avian and human H2N2 viruses

Summary The characteristics of an avian influenza virus were compared in detail with those of human Asian (H2N2) influenza viruses. Antigenic analysis by different antisera against H2N2 viruses and monoclonal antibodies to both the hemagglutinin and neuraminidase antigens showed that an avian isolate, A/duck/München/9/79 contained hemagglutinin and neuraminidase subunits closely related to those of the early human H2N2 viruses which had been prevalent in 1957. However, this avian virus gave low HI titers with absorbed and non-absorbed antisera to different human H2N2 viruses isolated in 1957. Like human Q phase variant, such as A/RI/5^–/57 (H2N2), hemagglutination of the above avian strain was not inhibited by the purified non-specific -inhibitor from guinea pig serum. Growth behavior at restrictive temperature (42° C) clearly differentiate the avian H2N2 virus from human influenza viruses, showing that the former virus grew well in MDCK cells at 42° C but not the latters. Genomic analysis of these viruses revealed that the oligonucleotide map of H2N2 virus isolated from a duck was quite different from those of human H2N2 viruses from 1957 to 1967. The oligonucleotide mapping also indicated that different H2N2 influenza virus variants had co-circulated in humans in 1957.With 2 Figures     

Analysis of mutations in the genome of cold-adapted strains of influenza A virus using extended modification of polymerase chain restriction method

Cold-adapted influenza viruses A/Leningrad/13 4/17/5 7 (H2N2) (Len/17) and A/Leningrad/I 34/4 7/57 (H2N2) (Len/47) are used in Russia to prepare live reassortant cold-adapted influenza vaccines (LIV) for adults and children, respectively. Comparison between the nucleotide sequences of the Len/17 strain and the initial wild-type strain A/Leningrad/13 4/5 7 (H2N2) revealed ten nucleotide substitutions (eight of them encoding). Four additional substitutions (three encoding) were found in the genome of the Len/47 virus. Gene segment restriction site (PCR-restriction) analysis was used for identification of the genotype of reassortant influenza viruses. Conventional methods of PCR-restriction analysis detect only five encoding nucleotides substitutions in the internal genes of the Len/17 and seven substitutions in the internal genes of the Len/47 virus. An extended modification of the PCR-restriction method detect all encoding mutations in the internal genes of the Len/17 and Len/47 viruses (eight and eleven encoding substitutions, respectively). This method is advantageous for genome composition analysis of reassortant influenza vaccine strains and for investigating the genetic stability of LIV during replication in vaccines.     

Accumulation of CD11b+Gr-1+ cells in the lung, blood and bone marrow of mice infected with highly pathogenic H5N1 and H1N1 influenza viruses

Infection with pathogenic influenza viruses is associated with intense inflammatory disease. Here, we investigated the innate immune response in mice infected with H5N1 A/Vietnam/1203/04 and with reassortant human H1N1 A/Texas/36/91 viruses containing the virulence genes hemagglutinin (HA), neuraminidase (NA) and NS1 of the 1918 pandemic virus. Inclusion of the 1918 HA and NA glycoproteins rendered a seasonal H1N1 virus capable of inducing an exacerbated host innate immune response similar to that observed for highly pathogenic A/Vietnam/1203/04 virus. Infection with 1918 HA/NA:Tx/91 and A/Vietnam/1203/04 were associated with severe lung pathology, increased cytokine and chemokine production, and significant immune cell changes, including the presence of CD11b⁺Gr-1⁺ cells in the blood, lung and bone marrow. Significant differential gene expression in the lung included pathways for cell death, apoptosis, production and response to reactive oxygen radicals, as well as arginine and proline metabolism and chemokines associated with monocyte and neutrophil/granulocyte accumulation and/or activation. Arginase was produced in the lung of animals infected with A/Vietnam/1204. These results demonstrate that the innate immune cell response results in the accumulation of CD11b⁺Gr-1⁺ cells and products that have previously been shown to contribute to T cell suppression.     

Generation and evaluation of an H9N1 influenza vaccine derived by reverse genetics that allows utilization of a DIVA strategy for control of H9N2 avian influenza

H9N2 avian influenza viruses have circulated widely in domestic poultry around the world, and their outbreaks have resulted in heavy morbidity and mortality. In addition, H9N2 avian influenza viruses were transmitted directly from birds to humans in Hong Kong and mainland China during 1998 and 2003, which prompted the public health authorities to seek protective strategies to control H9N2 influenza viruses. In this study, we attempted to develop a DIVA (differentiating infected and vaccinated animals) strategy for H9N2 avian influenza viruses. This strategy does not interfere with serological monitoring and allows effective control of H9N2 avian influenza. We generated a reassortant H9N1 influenza vaccine strain by reverse genetics and employed an enzyme-linked immunosorbent assay (ELISA) with a truncated N1 antigen expressed in E. coli to differentiate between vaccinated and naturally infected animals. Immunization of BALB/c mice with the inactivated reassortant H9N1 vaccine conferred protection against lethal challenge with H9N2 viruses. Meanwhile, the ELISA can be used to distinguish between vaccination and natural infection quickly and easily. Therefore, this study has opened up a new avenue for the control of H9N2 avian influenza.     

Restriction analysis of genome composition of live influenza vaccine

Live attenuated cold-adapted influenza vaccine (LIV) has been used in Russia for over 50 years and proved to be safe and effective. Currently, Russian reassortant LAIV is based on influenza AILeningrad/134/17/57 (H2N2) and B/USSR/60/69 Master Donor Viruses (MDVs) which are cold-adapted (ca), temperature-sensitive (ts), and attenuated (att), respectively. The MDVs are used to generate attenuated reassortant vaccine viruses containing the surface antigens of current wild type (wt) influenza A (HINI) and A (H3N2) viruses and wt influenza B virus. The ca/ts/att phenotype of these viruses limits replication in the upper respiratory tract. Reassortment typically yields numerous viruses with different genome constellations, rapid screening is needed to select proper vaccine viruses. In this study, screening of reassortant vaccine strains for live attenuated influenza vaccine generated from currently circulating influenza A and B viruses by RFLP assay is described.     

Extrapulmonary tissue responses in cynomolgus macaques (Macaca fascicularis) infected with highly pathogenic avian influenza A (H5N1) virus

The mechanisms responsible for virulence of influenza viruses in humans remain poorly understood. A prevailing hypothesis is that the highly pathogenic virus isolates cause a severe cytokinemia precipitating acute respiratory distress syndrome and multiple organ dysfunction syndrome. Cynomolgus macaques (Macaca fascicularis) infected with a human highly pathogenic avian influenza (HPAI) H5N1 virus isolate (A/Vietnam/1203/2004) or reassortants of human influenza virus A/Texas/36/91 (H1N1) containing genes from the 1918 pandemic influenza A (H1N1) virus developed severe pneumonia within 24 h postinfection. However, virus spread beyond the lungs was only detected in the H5N1 group, and signs of extrapulmonary tissue reactions, including microglia activation and sustained up-regulation of inflammatory markers, most notably hypoxia inducible factor-1α (HIF-1α), were largely limited to this group. Extrapulmonary pathology may thus contribute to the morbidities induced by H5N1 viruses.     

Production of early cytokines after infection with A/Leningrad/134/57 (H2N2) wide-type virus and cold-adapted attenuated vaccine viruses

The production of proinflammatory cytokines was studied following experimental infection of BALB/c mice with influenza viruses that differed in virulence. The generation of TNF-alpha, IL-6, IL-12, and IFN-gamma was investigated in the lung homogenates in the early periods after intranasal infection of mice with A/Leningrad/134/57 (H2N2) wild-type virus and cold-adapted attenuated vaccine viruses: A/Leningrad/134/17157 (H2N2) and A/Leningrad/134/47/57 (H2N2). Wild-type virus induced substantially higher levels of proinflammatory cytokines: TNF-alpha, IL-6, IL-12, and IFN-gamma. After infection with the cold-adapted viruses, the levels of the cytokines were reduced as compared to those induced by the wild-type virus. The A/Leningrad/134/47/57 virus was marked by a noticeable production of IL-6 and IFN-gamma in the murine lung, but it was less than with wild-type virus infection. At the same time, the more attenuated strain A/Leningrad/134/47/57 induced TNF-alpha and IFN-gamma in the quantities similar to those in the control animals. Thus, a response of proinflammatory cytokines in early infection in the murine lung depended on the level of viral replication in the lower respiratory tract and on the attenuation of influenza virus strains.     

Stimulation of homo- and heterologic T-cell immunological memory in volunteers inoculated with live influenza A (H5N2) reassortant vaccine

The study deals with the ability of live attenuated reassortant influenza vaccine (LAIV) A (H5N2) to stimulate a CD4+ and CD8+ immunological memory T cell-mediated immune response in volunteers. These data were compared with the quantitative characteristics of a humoral immune response. A two-dose regimen of intranasal vaccination of avian influenza na?ve people with A (H5N2) LAIV induced the production of circulating CD4+ and CD8+ memory cells specific to both A (H5N2) and seasonal A (H1N1) influenza strains. Some of the volunteers were not absolutely A (H5N2) influenza virus na?ve since they had been found to have this virus-specific cross-reactive immunological memory T-cells in the prevaccination period. The content (%) of these cells varied significantly within the group. The quantitative values of postvaccination CD4+ and CD8+ memory cell accumulation were inversely related to their prevaccination level.