Cold-adapted Influenza Virus Research Articles

Detection of cold-adapted vaccine-strain influenza virus using two commercial assays.

Because of the contagious nature of influenza virus it is necessary to identify infected individuals after the virus is introduced into a population. The aim of this study was to characterise influenza virus detection with commercially available assays after intranasal vaccinating horses with cold-adapted influenza virus. Seven horses were vaccinated and placed with 3 unvaccinated horses. Nasal secretion samples were evaluated using 2 antigen detection assays. All 10 horses were positive in the Flu OIA assay during the study period, but only one horse was positive on one sample using the Directigen Flu A assay. Horses were most likely to be positive during the first 3 days following vaccination, and several horses were intermittently positive for several days after this. Obtaining positive test results from nonvaccinated, incontact horses suggests they became infected with vaccine-strain virus that was shed by vaccinated horses. These results are important for the correct interpretation of influenza antigen detection tests in situations when this modified-live intranasal vaccine has been used.

Stabilizing cold-adapted influenza virus vaccine under various storage conditions

Various diluents, stabilizers, buffers, and storage conditions were assessed for their efficacy in stabilizing cold-adapted influenza virus vaccine. Frozen liquid vaccine formulations, comprised of a normal uninfected allantoic fluid diluent and an SPG (sucrose–phosphate–glutamate) stabilizer, generated complete stability of H1N1, H3N2, and Type B strains for at least 1 year of storage at −20 °C. The ability to store live influenza virus frozen liquid vaccines, at the moderate temperature of −20 °C, has not been demonstrated previously. This significant advance could facilitate influenza vaccine storage and administration in the clinic, and subsequently increase marketability. The stability of lyophilized formulations was also augmented by the addition of 2% Casitone and the control of pH with 0.066 M phosphate in the SPG stabilizer. This alternative formulation may be useful in markets where freezing is not feasible or short-term room temperature storage is necessary.

Cold-attenuated live influenza vaccines, a risk-benefit assessment

The principle of live attenuated influenza vaccines has been known for many decades. However, the pharmaceutical and clinical development according to current regulations, of modern live influenza vaccines based on cold adapted influenza viruses (CAIV) started only recently and these vaccines will most probably become an alternative within the next couple of years to licensed inactivated influenza vaccines that have been used routinely since the early 1940s. In contrast to contemporary trivalent inactivated influenza vaccines, which are administered intramuscularly, trivalent CAIV-based vaccines will be administered intranasally as a spray. Quality, safety and efficacy aspects related to CAIV vaccines as well as possible risks linked to the widespread use of these vaccines will be discussed in the following overview and compared to established influenza vaccines. Moreover, issues of practicality of CAIV vaccines focusing on the necessity of an annual update of influenza vaccines are addressed.

Intranasal Administration of Influenza Vaccines

AbstractThis review article focuses on intranasal immunisation against influenza,although it also encompasses antigen uptake and processing in the nasopharyngealpassages, host defence from influenza and current influenza vaccination practices.Improvement of current vaccination strategies is clearly required; current proceduresinvolve repeated annual injections that sometimes fail to protect the recipient. It isenvisaged that nonpercutaneous immunisation would be more attractive to potentialvaccinees, thus improving uptake and coverage. As well as satisfying noninvasivecriteria, intranasal influenza immunisation has a number of perceived immunologicaladvantages over current procedures. Perhaps one of the greatest attributes of thisapproach is its potential to evoke the secretion of haemagglutinin-specific IgAantibodies in the upper respiratory tract, the main site of viral infection. Inactivated influenza vaccines have the advantage that they have a long historyof good tolerability as injected immunogens, and in this respect are possibly morelikely to be licensed than attenuated viruses. Inert influenza vaccines are poormucosal immunogens, requiring several administrations, or prior immunologicalpriming, in order to engender significant antibody responses. The use of vaccinedelivery systems or mucosal adjuvants serves to appreciably improve theimmunogenicity of mucosally applied inactivated influenza vaccines. As is the casewhen they are introduced parenterally, inactivated influenza vaccines are relativelypoor stimulators of virus-specific cytotoxic T lymphocyte activity following nasalinoculation. Live attenuated intranasal influenza vaccines are at a far moreadvanced stage of clinical readiness (phase III versus phase I). With the use of liveattenuated vaccines, it is possible to stimulate mucosal and cell-mediatedimmunological responses of a similar kind to those elicited by natural influenzainfection. In children, recombinant live attenuated cold-adapted influenza viruses arewell tolerated. Moreover, cold-adapted influenza viruses usually stimulate protectiveimmunity following only a single nasal inoculation. Safety of recombinant liveattenuated cold-adapted influenza viruses has also been demonstrated in high riskindividuals with cystic fibrosis, asthma, cardiovascular disease and diabetes mellitus.They are not suitable for immunising immunocompromised patients, however, andare poorly efficacious in individuals with pre-existing immunity to strains closelyantigenically matched with the recombinant virus. According to the reviewedliterature, it is apparent that intranasal administration of vaccine as an aerosol issuperior to administration as nose drops. The information reviewed in this papersuggests that nasally administered influenza vaccines could make a substantialimpact on the human and economic cost of influenza.

The development of live attenuated cold-adapted influenza virus vaccine for humans.

A procedure to attenuate live influenza virus of type A and type B was developed using adaptation of the virus to grow at 25 degrees C (cold adaptation; ca). Through a series of stepwise passages, two stable mutants were obtained and designated as 'Master' strains, one for type A influenza virus (A/Ann Arbor/6/60-H2N2) and one for type B influenza virus (B/Ann Arbor/1/66). These mutants were used in genetic reassortment using either the classical method or more recently described reverse genetics to update the relevant surface antigens of the circulating strains of influenza virus. The derivation is based on the concept of 6/2 where 6 signifies the six internal genes of the master strain and 2 refers to the two genes coding for the two surface glycoproteins HA and NA of the circulating influenza virus. The advantages of this vaccine were demonstrated to be (1) proper level of attenuation, (2) non-transmissibility, (3) genetic stability, (4) presence of the ca and ts markers and (5) immunogenicity involving both local and the cell-mediated immune responses. The clinical trials in infants, children, adults and elderly have provided the necessary data for eventual licensing of this vaccine. The ease of administration (intranasal) safety and high efficacy make this vaccine suitable to prevent influenza virus infection in all age groups.

Factors affecting the yield of cold-adapted influenza virus vaccine

Various manipulations to production procedures have been investigated in order to discover methods to attain adequate or augmented titers of cold-adapted influenza virus (CAIV) vaccine. The methods modified include those used for reassortant selection and the determination of virus growth parameters. Increased infectivity titers were achieved through selection of high-yielding mutants by isolating multiple plaques during plaque purification of reassortant clones, as well as through optimiztion of egg incubation times, age, and lot for individual strains. Up to 6-fold increases in virus yield were obtained by selecting high yielding mutants, up to 9-fold increases were achieved by modifying egg incubation times, and a nearly 1 log increase was realized by determining the ideal egg age for individual strains.

Cold-adapted live attenuated influenza vaccines developed in Russia: can they contribute to meeting the needs for influenza control in other countries?

It is now more than 30 years since the first cold-adapted influenza viruses were developed in Russia as potential live, attenuated vaccines. In the past 15-20 years considerable experience has been gained from Russian and joint Russian-US laboratory and clinical studies with type A monovalent and bivalent vaccines prepared with genetic reassortant viruses derived from one of these cold-adapted viruses in particular. A/Leningrad/134/57. More recent experiences include use of trivalent cold-adapted vaccines with a type B component. The overall high level of safety of individual and combined vaccines in pre-school and school-aged children, with illness reductions in open field trials equivalent to that seen with inactivated vaccines, is such as to suggest that practical measures might now be justified to facilitate expansion of the use of these vaccines to other countries. It is proposed that further experimentation with the Russian cold-adapted live attenuated vaccines should be focused on issues that will relate to the public health perspective, i.e. selection of the single best candidate type A and B vaccines for intense study using as criteria their potential for meeting licensing requirements outside Russia, and documenting the clinical protective efficacy of a single vaccine dose compared to two doses as studied until now. Resolution of these issues is important to ensure that costs for future live vaccine production, control, and utilization will be kept at lowest levels so that expanded use of live vaccines will have maximum cost-benefit and afford-ability. To guide those interested in these issues, examples are given of populations for whom a licensed live cold-adapted vaccine might be considered, together with indications of extra data needed to fully validate each suggested use.

Prevention of influenza by the intranasal administration of cold-recombinant, live-attenuated influenza virus vaccine: importance of interferon-γ production and local IgA response

To clarify which immunological factors were more effective in preventing influenza virus infection, we measured immunological parameters induced by vaccination and infection in vivo and in vitro. Healthy adult subjects (n = 128) were divided into vaccinated (n = 85) and untreated (n = 43) groups. Eighty-five were vaccinated intranasally with a trivalent cold-adapted recombinant influenza virus vaccine containing type A (H1N1 and H3N2) and B viruses. Subjects were mostly seropositive before vaccination. In 29 (80.6%) of the 36 examinees showing a prevaccination HI antibody titre of less than 1:128, the titre increased more than four times after vaccination. On the other hand, an increase of more than four times was found in four (8.2%) of the 49 individuals who had shown a prevaccination titre of more than 1:128. The IgA antibody was negligibly detected in the nasal wash specimens before vaccination, and was induced by vaccination in some cases. Lymphocyte proliferation and interleukin 2 (IL-2) production in cultured lymphocytes of the same subjects stimulated by H1N1 virus in vitro were correlated with the HI antibody titre. However, the interferon γ (IFN-γ) production was low before vaccination, regardless of the HI antibody titre, and showed a significant increase after vaccination. It was suggested that local IgA response and IFN-γ production play important roles in the prevention of influenza. Since there was the outbreak of influenza A (H1N1) in Kochi Prefecture after completion of blood samples 6–8 weeks after the second vaccination, we examined the above hypothesis. A significantly (p<0.01) lower morbidity rate in the vaccinated examinees was found. The HI antibody, lymphocyte DNA synthesis and IL-2 production, which had been increased by vaccination, showed little further increase in response to the epidemic. In contrast, local IgA response and IFN-γ production, which had been increased by vaccination, showed another increase because of the epidemc.

Variability in Infectivity of Cold-Adapted Recombinant Influenza Virus Vaccines in Humans

Journal Article Variability in Infectivity of Cold-Adapted Recombinant Influenza Virus Vaccines in Humans Get access Wendy A. Keitel, Wendy A. Keitel Reprints or correspondence: Dr. Wendy A. Keitel, Acute Viral Respiratory Disease Unit, Baylor College of Medicine, One Baylor Plaza, Houston. TX 77030. Search for other works by this author on: Oxford Academic PubMed Google Scholar Robert B. Couch, Robert B. Couch Search for other works by this author on: Oxford Academic PubMed Google Scholar Thomas R. Cate, Thomas R. Cate Search for other works by this author on: Oxford Academic PubMed Google Scholar Hunein F. Maassab Hunein F. Maassab Search for other works by this author on: Oxford Academic PubMed Google Scholar The Journal of Infectious Diseases, Volume 169, Issue 2, February 1994, Page 477, https://doi.org/10.1093/infdis/169.2.477 Published: 01 February 1994

Developments in mucosal influenza virus vaccines

Immunogenicity and efficacy of aerosol inactivated split influenza virus vaccines, which are threefold the strength of the vaccines for parenteral use, and cold-adapted reassortant live influenza virus vaccines were evaluated. Mucosal immune responses were evaluated by quantifying specific IgA antibody of the nasal swab solution, and systemic immune responses were evaluated by determining serum haemagglutination inhibition antibody levels. Efficacy of the aerosol inactivated vaccine was evaluated by a challenge test using live vaccine virus. It was concluded that mucosally administered inactivated influenza virus vaccine stimulated systemic and mucosal immune responses more strongly than live influenza virus vaccine and manifested a much stronger booster effect than live vaccine. Mucosal administration of inactivated influenza virus vaccine was effective in preventing infection by live vaccine virus.

Trivalent Attenuated Cold-Adapted Influenza Virus Vaccine: Reduced Viral Shedding and Serum Antibody Responses in Susceptible Adults

Trivalent cold-adapted recombinant (CR) influenza virus vaccines containing types A (H1N1 and H3N2) and B viruses were evaluated in two double-blind, placebo-controlled trials. Susceptible adults were randomly assigned to receive the following vaccines by intranasal drops 1 month apart: two doses of trivalent vaccine, bivalent CR influenza A (Bi A) vaccine followed by monovalent B (Mono B) vaccine or vice versa, or two doses of placebo. All vaccines were well tolerated. Shedding of each of the three vaccine viruses was reduced after the first dose of trivalent vaccine compared with primary vaccination with Bi A or Mono B. Shedding was also reduced after second vaccinations, whether homologous (trivalent-trivalent) or heterologous (Bi A/Mono B or Mono B/Bi A). Reduced viral shedding was associated with reduced serum antibody responses. Thus, both simultaneous and sequential inoculations of susceptible adults with CR influenza vaccine viruses result in reduced viral shedding and serum antibody responses.

SAFETY AND IMMUNOGENICITY OF LIVE ATTENUATED INFLUENZA VACCINES IN INFANTS AND CHILDREN

Inactivated influenza vaccines confer incomplete and transient protection from disease, and the whole-virus inactivated vaccines cause high rates of reactions in children. Live, attenuated vaccines have been developed from cold-adapted (CA) and avian-human (AH) influenza virus reassortants, and are safe and effective in adults. We compared the infectious dose50, immunogenicity, reactogenicity and transmissability of CA and AH influenza A/Bethesda/85 (H3N2) reassortant vaccines in young children. Fifty-eight seronegative 6 to 36 months old children were studied in groups of 6 to 10 in close contact in a day-care-like setting for 2 days before and 9 days after receiving a placebo or 103 to 106 TCID50 of either vaccine. Seroconversion occurred in >50% of children who received a dose of 106 TCID50 of either vaccine. There were no differences in rates of febrile or respiratory illness between vaccinees and placebo recipients. Vaccine virus was shed in low titers (5.6 to 27.5 TCID50/ml) for 1 to 5 days by vaccinees, and no transmission to placebo contacts occurred. Both vaccines appear to be safe and immunogenic in young children and infants.

Comparative studies of wild-type and cold-mutant (temperature-sensitive) influenza virus: detection of mutations in all genes of the A/Ann Arbor/6/60(H2N2) mutant vaccine donor strain

Direct biochemical evidence has been obtained for the existence of mutations in all eight RNA segments of the A/Ann Arbor/6/60 cold-adapted (ca) mutant influenza virus strain as compared with its wild-type (wt) progenitor. Polyacrylamide gel electrophoresis (PAGE) of viral RNA revealed a change in the electrophoretic migration of RNA 2 (PB1). T 1 oligonucleotide mapping revealed changes in two polymerase genes (the PB2 and PA genes), the hemagglutinin (HA) gene and the nucleoprotein (NP) gene. Analysis of S 1 nuclease-treated RNA hybrids on polyacrylamide gels detected changes in the HA and neuraminidase (NA) genes. Partial DNA sequence analysis demonstrated a base sequence change in the matrix (M) protein gene that predicts an amino acid change in the M 2 protein and a silent mutation in the non-structural (NS) protein gene. In addition, analysis of viral polypeptides by PAGE has so far revealed changes in the viral protein, PA. These findings directly demonstrate the existence of multiple mutations in the ca vaccine strain, a property that may provide reliably and stably attenuated vaccines that derive their six internal genes from the ca A/Ann Arbor/6/60 donor strain.

Serological evaluation of an influenza A virus cold-adapted reassortant live vaccine, CR-37 (H1N1), in Japanese adult volunteers.

A cold-adapted influenza A virus, CR-37 (H1N1), derived from genetic reassortment between A/Ann Arbor/6/60 (H2N2) cold-adapted variant virus and A/California/10/78 (H1N1) wild-type virus, was tested in Japanese adult volunteer. The CR-37 live virus preparation induced only low-grade clinical reactions in volunteers for the first 3-4 days after inoculation. Two vaccinees who did not show any antibody changes became febrile (over 38.0 degrees C). Skin tests using the vaccine preparation and uninfected allantoic fluid were performed, and indicated that one of these two vaccines was positive for the CR-37 vaccine preparation. A high proportion of the vaccinees whose sera had a haemagglutination-inhibition (HI) antibody titre against the vaccine strain of less than or equal to 64 before inoculation, seroconverted in both HI and neuraminidase-inhibition (NAI) antibody titrations, and only a few seroconverted in the titration of antibody against type-specific internal antigens. The serological examinations against heterotypic H1N1 variants indicated that the cold-adapted live influenza virus vaccine could induce a broad spectrum of HI antibody reactivity and immunity of long duration.

ADVANTAGE OF LIVE ATTENUATED COLD-ADAPTED INFLUENZA A VIRUS OVER INACTIVATED VACCINE FOR A/WASHINGTON/80 (H3N2) WILD-TYPE VIRUS INFECTION

The efficacy of live attenuated cold-adapted (ca) reassortant influenza virus vaccine against experimental challenge with homologous wild-type virus 5 to 8 weeks after vaccination was compared with that of licensed inactivated vaccine in 81 seronegative (haemagglutination-inhibition antibody titre 1:8) college students. At a dose of 107·5 50% tissue culture infectious dose (TCID50) (70 HID50, human 50% infectious doses) the live virus vaccine, given intranasally, completely protected against illness caused by wild-type virus, whereas the inactivated vaccine, administered intramuscularly, provided 72% protection. Wild-type virus was recovered from only 13% of live virus vaccinees (107·5 TCID50 dose of ca virus) compared with 63% of inactivated virus vaccinees and the few infected live virus vaccinees shed 1000 times less wild-type virus than did infected inactivated virus vaccinees or unvaccinated controls. This striking reduction in virus shedding suggests that influenza transmission may be more efficiently interrupted with live than with inactivated virus vaccination.

Genetic stability of A/Ann Arbor/6/60 cold-mutant (temperature-sensitive) live influenza virus genes: analysis by oligonucleotide mapping of recombinant vaccine strains before and after replication in volunteers.

Ten cold-adapted (ca) recombinant influenza viruses that had been derived by reassortment between the ca temperature-sensitive (ts) A/Ann Arbor/6/60 mutant and selected wild-type viruses were examined by RNA oligonucleotide mapping for the presence or absence of several previously identified oligonucleotide markers. These markers comprise oligonucleotides that appeared only in the mutant donor strain after cold adaptation and other oligonucleotides present only in the parent A/Ann Arbor/6/60 wild-type virus. The markers in the mutant donor strain were transferred without change to the recombinant ca vaccine strains, and reversion causing a reappearance of markers for the wild-type A/Ann Arbor/6/60 virus did not occur. Furthermore, examination by oligonucleotide mapping of 10 isolates from vaccinated, seronegative children revealed a high degree of genetic stability of the viral genome; an oligonucleotide change was detected in only one of the 10 isolates examined.

Relative immunogenicity of the cold-adapted influenza virus A/Ann Arbor/6/60 (A/AA/6/60-ca), recombinants of A/AA/6/60-ca, and parental strains with similar surface antigens

The immunogenicity of several cold-adapted (ca) viruses was compared in CSL mice with that of wild-type parental viruses with similar surface antigens, according to the vaccinating dose required to clear a challenge consisting of 10(4.5) 50% tissue culture infective doses of the wild-type virus. All ca viruses were less immunogenic than their wild-type parental strains by a factor of 10(1.3) to 10(3.4), probably due to the restricted capacity of ca viruses to replicate in the respiratory tracts of mice. However, their immunogenicity was considerably enhanced when two quite small doses were administered 3 weeks apart. The immunogenicity of ca viruses when administered in two doses and wild-type viruses when administered as a single dose varied according to their surface antigens. It was highest for viruses with the H2N2 A/Ann Arbor/6/60 and H3N2 A/Queensland/6/72 surface antigens and lowest for those with H1N1 A/HK/123/77 surface antigens. When two doses consisting of 10(5.0) 50% tissue culture infective doses of A/Ann Arbor/6/60-ca were administered at an interval of 3 weeks, solid immunity was induced against the wild-type A/Ann Arbor/6/60 parental virus, two heterologous H3N2 strains, and an H1N1 strain.

In vitro production of anti-influenza virus antibody after intranasal inoculation with cold-adapted influenza virus.

We have studied the production of anti-influenza virus antibody in vitro by peripheral blood mononuclear cells (PBMC) obtained from 7 normal volunteers at various times after intranasal inoculation with cold-adapted A/Alaska/6/77 [H3N2] influenza virus. Antibody released into culture supernatants was assayed by a 2-step enzyme-linked immunosorbent assay (ELISA). Cells obtained 6 days after intranasal inoculation spontaneously released both IgG and IgA anti-influenza antibody; this antibody production occurred within 24 hr, was specific for the virus used to inoculate the volunteers, and was inhibitable by cycloheximide. When day 6 cells were cultured in vitro for 12 days with the polyclonal activator pokeweed mitogen (PWM), no increase in the amount of antibody above that released in the absence of PWM was seen. In contrast, cells obtained 27 days after inoculation made no spontaneous antibody. In addition, cultures of these day 27 cells with PWM resulted in the production of large amounts of IgG antibody but relatively little IgA anti-virus antibody. Thus, after a mucosal influenza virus infection, several subpopulations of functionally different cells sequentially appear in the peripheral circulation: an initial population of cells secreting IgG and IgA antibody spontaneously, followed by a 2nd population of cells secreting IgG antibody when stimulated with PWM.

Growth of influenza A viruses in hamsters.

The growth characteristics of four temperature-sensitive or cold-adapted recombinant influenza viruses and eight recombinant influenza viruses derived in other ways, together with the wild-type, parent viruses of these strains, were tested in hamster lungs and turbinates and in embryonated eggs at different temperatures for their replicative ability. The results showed that although the temperature-sensitive and cold-adapted recombinant viruses replicated to considerably lower titres than their wild-type parent virus strains in hamster lung and at 37 degrees C in embryonated eggs, no similar pattern of growth was observed for the group of A/PR 8 and A/Okuda recombinant influenza viruses studied in these systems. The hamster model is not therefore generally applicable as a marker for attenuated influenza virus vaccine strains.

Biologic and Immunologic Characteristics of Cold-Adapted Influenza Virus

Summary Cold variants of influenza virus showed an impaired capacity to reproduce at acid pH (5.7 to 6.3) and elevated temperature (41°C). These features were used as markers to differentiate the cold variant from the original “wild” strain. After eight consecutive passages in mice, the viral strains recovered from the lungs of infected mice could still be identified by these selective markers as the cold variants. They showed no reversion to virulence upon ten consecutive passages in mice and retained their capacity to elicit a good antibody response. Ferrets responded as did mice to infection with the cold variants; the animals showed no evidence of disease and developed good HI antibody response. In addition, ferrets that possessed circulating antibody showed a heightened antibody response after they were reinoculated intranasally with cold variant virus.