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PubMed2002

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 degrees C. The ability to store live influenza virus frozen liquid vaccines, at the moderate temperature of -20 degrees 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.

Adaptation, Physiological↗
PubMed1986

Heterogeneity and structure of brain tubulins from cold-adapted Antarctic fishes. Comparison to brain tubulins from a temperate fish and a mammal.

Tubulins purified from brain tissue of Antarctic fishes assemble in vitro to form microtubules at the low temperatures experienced by these extreme psychrophiles (Williams, R. C., Jr., Correia, J. J., and DeVries, A. L. (1985) Biochemistry 24, 2790-2798). We have initiated studies to determine the structural requirements for assembly of Antarctic fish tubulins at low temperatures. As a first step we have compared the heterogeneity, structures, amino acid compositions, and net charge of brain tubulins purified from three Antarctic fishes (Notothenia gibberifrons, Notothenia coriiceps neglecta, and Chaenocephalus aceratus), from the temperate channel catfish (Ictalurus punctatus), and from a mammal (the cow). Each preparation contained the alpha- and beta-tubulins and was free of microtubule-associated proteins. When examined by isoelectric focusing and by two-dimensional electrophoresis, brain tubulins from the Antarctic fishes were found to be highly heterogeneous; each was resolved into approximately 20 isoelectric variants. The distributions of the isotubulins from the cold-adapted fishes were similar but differed significantly from those of tubulins from catfish and cow. The average isoelectric points of the alpha- and beta-tubulins from the Antarctic fishes were more basic than the isoelectric points of the corresponding tubulins from bovine brain. Peptide mapping confirmed that tubulins from the Antarctic fishes and the mammal differed in structure. The amino acid compositions of fish and mammalian tubulins were similar, but Antarctic fish tubulins apparently contained fewer Glx residues than did catfish or bovine tubulins. Finally, native tubulins from an Antarctic fish and the cow differed slightly in net negative charge. Thus, brain tubulins from the cold-adapted fishes differ structurally from the tubulins of a temperate fish and of a mammal.

Acclimatization↗
PubMed2004

Cell-based assay for the determination of temperature sensitive and cold adapted phenotypes of influenza viruses.

The determination of temperature sensitive (ts) and cold adapted (ca) phenotype for influenza A and B strains has been conducted traditionally using embryonated chicken eggs. As attempts are made to move away from the use of eggs in the manufacturing process of influenza vaccines, it will become useful to develop cell-based assays to support cell culture-based vaccine production. In this study, MDCK cells have been evaluated as a tool for determining the ts and ca phenotypes associated with live attenuated influenza viruses. Direct comparisons were made of these phenotypes carried out in eggs. Reassortants made from the Russian live attenuated influenza donor strains A/Leningrad/134/17/57 (H2N2) and B/USSR/60/69 were prepared entirely in MDCK cells and their phenotypes evaluated using the MDCK cell-based assay. It is concluded that MDCK cells are more sensitive than eggs for the measurement of ts and ca phenotype of influenza viruses (particularly for influenza A) and they provide an alternative means for screening candidate reassortants prior to determining their genome composition.

Animals↗
PubMed1993

Immunization of institutionalized asthmatic children and patients with psychomotor retardation using live attenuated cold-adapted reassortment influenza A H1N1, H3N2 and B vaccines.

Live attenuated cold-adapted reassortant (CR) influenza virus vaccines were evaluated in institutionalized asthmatic children and severe psychomotor-retarded (SPR) patients. Almost all the vaccinees were seropositive to the vaccine strains before immunization. Trivalent CR vaccine (containing A H1N1 (CR-125), A H3N2 (CR-149) and B (CRB-117)), bivalent CR vaccine (CR-125 and CR-149) and monovalent CRB-117 were inoculated to 19 asthmatic children and 36 and 16 SPR patients, respectively. Overall 49, 22, and 11% of vaccinees were infected by A H1N1, A H3N2 or B vaccine viruses, respectively, as indicated by significant haemagglutination-inhibition (HI) antibody titre rises 4 weeks after inoculation. No severe adverse reactions associated with CR vaccination were observed in the handicapped patients. A nosocomial outbreak of influenza A H1N1 occurred in the ward with asthmatic children, but none of the 19 CR-trivalent vaccinees became infected. However, five of 20 non-vaccinees in the same ward, and ten of 30 vaccinees in another ward that received inactivated split vaccine became infected. The CR vaccines demonstrated significant protective effects against natural exposure to the A H1N1 virus, and were well tolerated and safe when given to patients with bronchial asthma and severe psychomotor retardation.

Adolescent↗
PubMed1990

Evaluation of the infectivity, immunogenicity, and efficacy of live cold-adapted influenza B/Ann Arbor/1/86 reassortant virus vaccine in adult volunteers.

A cold-adapted (ca) influenza B reassortant that derived two genes encoding the hemagglutinin and neuraminidase from influenza B/Ann Arbor/1/86 wild-type virus and six internal RNA segments from ca influenza B/Ann Arbor/1/66 virus was evaluated in 66 adult volunteers having a serum hemagglutination inhibition antibody titer less than or equal to 1:8. The ca reassortant was attenuated and elicited the production of systemic and local antibodies; the 50% human infectious dose was 10(6.4) TCID50. Six weeks after vaccination, 12 unvaccinated volunteers and 13 recipients of ca virus (10(7.5) TCID50) were challenged experimentally with homologous wild-type influenza B virus. The ca vaccine completely protected against illness, and the magnitude of shedding was 50-fold less in vaccinees than in unimmunized controls, five of whom became ill. These findings indicate that the six internal RNA segments of the ca influenza B/Ann Arbor/66 donor virus confer desirable properties of a live virus vaccine to a reassortant derived from a virulent virus. Such reassortants may be suitable vaccines for healthy adults.

Adult↗
PubMed1988

[Role of the paraventricular nuclei of the hypothalamus in the development of cold adaptation in albino rats].

The role of paraventricular hypothalamic nuclei (PVN) in the development of cold adaptation was studied in chronic experiments on white rats. Animals with the PVN lesions revealed a low resistance against cooling, a small increase of brown adipose tissue mass and no calorigenic effect of noradrenaline after long-lasting cold exposure. The data obtained suggest that the PVN lesions delayed the development of specific mechanisms of adaptation to low temperature.

Adaptation, Physiological↗
PubMed1993

A cold-adapted mutant of parainfluenza virus type 3 is attenuated and protective in chimpanzees.

A live attenuated cold-adapted parainfluenza virus type 3 (PIV-3) vaccine is being developed to prevent the serious lower respiratory tract disease caused by this virus in infants and young children. This cold-passaged mutant (cp45) was evaluated in seronegative chimpanzees and found to be highly attenuated in both the upper and lower respiratory tracts compared to its wild-type parent. Animals immunized with cp45 were also highly resistant to wild-type PIV-3 challenge. Stability of the attenuation phenotype was demonstrated by the administration to 2 additional chimpanzees of an isolate of cp45 obtained after 10 days of replication in a chimpanzee. It was attenuated in both the upper and lower respiratory tracts. The cp45 virus present in the respiratory tract secretions of chimpanzees retained the temperature-sensitive (ts) phenotype, but some loss of ts property was observed in the isolates. These results provide a basis on which to proceed to clinical trials in seronegative human infants and children.

Adaptation, Physiological↗
PubMed1991

Safety and immunogenicity of live attenuated cold-adapted influenza B/Ann Arbor/1/86 reassortant virus vaccine in infants and children.

A cold-adapted (ca) influenza B reassortant vaccine consisting of two genes encoding the hemagglutinin and neuraminidase from wild-type influenza B/Ann Arbor/1/86 virus and the six internal RNA segments from influenza B/Ann Arbor/1/66 ca virus was evaluated in 18 seropositive and 57 seronegative infants and children. The ca reassortant was infectious in seronegative vaccinees, with an estimated 50% human infectious dose of 10(2.5) TCID50. Nasal wash specimens from vaccinees retained the temperature-sensitive phenotype, indicating that the virus was phenotypically stable after replication in fully susceptible children. The vaccine was highly immunogenic in the seronegative vaccinees; 54% of the seropositive vaccinees also developed an increase in serum antibody. The ca vaccine was well tolerated, with only a mild increase in upper respiratory tract symptoms seen in the seronegative vaccinees. These studies indicate that the B/Ann Arbor/1/86 ca reassortant is safe, immunogenic, and phenotypically stable in infants and children.

Antibodies, Viral↗
PubMed1979

[Changes in muscular thermogenesis in cold adapted rats following beta-adrenoreceptor blockade].

The administration of beta-adrenergic blocking agent propranolol to cold adapted rats entailed some decrease of the total metabolic reaction and body temperature as well as an additive (compensatory) increase of electrical muscle activity. The compensatory effect was more obvious in postural-tonic groups of muscles (m. trapezius, m. masseter) and in deeper portions of muscles (m. tibialis ant., m. trapezius), mainly presented by the red fibers. An adaptive increase of non-shivering thermogenesis and temperature effect of muscular contraction seem to be mainly controlled by beta-adrenergic mechanisms.

Adaptation, Physiological↗
PubMed1994

Dominance of cold-adapted influenza A virus over wild-type viruses is at the level of RNA synthesis.

Cold-adapted (ca) influenza A virus is dominant over wild-type (wt) influenza A viruses in mixed infections of MDCK cells. Since the inhibition of the growth of wt viruses occurs at or before the level of protein synthesis, the effect of coinfection by ca virus on RNA synthesis of wt viruses was investigated. RNA from single and mixed infections of ca and wt viruses was analyzed by hybridization with positive and negative sense oligonucleotide probes capable of distinguishing the RNAs of the two viruses. Primary and secondary transcription of mRNA and replication of vRNA from an early (NP) and a late (M) gene were quantitated. Although all stages of RNA synthesis were reduced, the key inhibition of wt RNA synthesis in coinfections with ca virus was at the level of vRNA replication. The inhibition of wt RNA synthesis occurred in mixed infections without any corresponding reduction of vRNA or mRNA synthesis by ca virus. Mechanisms by which ca virus may inhibit wt virus RNA synthesis are proposed based on the role of the products of gene segment 7 of the ca virus, the gene known to be responsible for the dominance phenotype.

Adaptation, Physiological↗
PubMed1980

Involvement of adrenergic receptors in skeletal muscle metabolism of the cold-adapted rat.

Hind-limb perfusion was used to investigate alterations of alpha and beta receptor-mediated metabolic effects in cold-adapted (CA) rats. The response to beta receptor stimulation by isoproterenol in the isolated hind-limbs of CA rats was slightly diminished. Oxygen consumption and lactate production were reduced in CA rats after beta receptor stimulation. Noradrenalin infusion caused less vasoconstriction in CA rats than in the controls (CO). Desensitization of alpha and beta receptors due to chronic sympathetic overstimulation may be the underlying cause of these observations. Compared with the controls, metabolism was enhanced in perfused hind-limbs of CA rats with an active nervous system. Decreased vascular resistance due to the lower perfusion pressure in CA rats might contribute to this increased metabolism.

Adaptation, Physiological↗
PubMedDate not supplied

[The cardiotropic, hypometabolic and hypothermic activity of peptide fractions from the tissues of hibernating cold-adapted animals].

From tissues of hibernating and active long-tailed ground squirrels and from the brain of cold-adapted Yakut horses, low molecular peptide fractions were obtained which, after injection to albino mice, decreased oxygen consumption and rectal temperature in them. The same fractions exhibited negative chrono- and inotropic effects on isolated hearts of ectothermic and endothermic animals. Fractions from the brain of ground squirrels and the brain of horse exhibited similar pattern of the activity. The activity of fractions was subjected to seasonal changes and depended on the degree of their purification. Provisional intracellular microelectrophysiological analysis of the effect of these fractions on the frequency and strength of contractions in isolated heart was made.

Adaptation, Physiological↗
PubMed1985

Response of seronegative and seropositive adult volunteers to live attenuated cold-adapted reassortant influenza A virus vaccine.

The infectivity and immunogenicity of live attenuated A/Washington/897/80 cold-adapted reassortant virus vaccine was evaluated in seronegative (hemagglutination inhibition titer, less than or equal to 1:4) and seropositive (hemagglutination inhibition titer, greater than 1:4) adult volunteers. The vaccine was efficient in infecting seronegative volunteers (94%). Moreover, 51% of seropositive vaccinees were infected by the virus. After live virus vaccination, greater than 83% of both seronegative and seropositive vaccinees achieved a level of nasal wash antibody previously associated with resistance to infection with influenza A virus. These findings indicate that both seronegative and seropositive vaccinees can benefit from live virus vaccination.

Adolescent↗
PubMed1997

Postcranial evidence of cold adaptation in European Neandertals.

The low brachial and crural indices of the European Neandertals have long been considered indicative of cold adaptation. Recent work has documented lower limb/trunk ratios and deeper chests (anterior-posterior diameter) in European Neandertals than among their successors. The present study uses variables reflective of limb length, body mass and trunk height, and compares European Neandertals to 15 globally diverse recent human samples (1 "Eskimo," 3 North African, 4 sub-Saharan African and 7 European). Bivariate plots, as well as principal components analysis plots of log shape-transformed data, indicate that European Neandertals had an overall body shape that falls at the extreme end of modern higher latitude groups' range of variation. Cluster analysis (minimum spanning tree on a principal coordinates plot) indicates that the Neandertals are closest in body shape to modern "Eskimos," but even in this dendrogram, they are joined to the "Eskimo" via a long branch. In fact, it appears that European Neandertals were "hyperpolar" in body shape, likely due to two factors: 1) the extremely cold temperatures of glacial Europe and 2) less effective cultural buffering against cold stress.

Adaptation, Biological↗
PubMed2003

An evaluation of the genetic stability and pathogenicity of the Russian cold-adapted influenza A donor strains A/Leningrad/134/17/57 and A/Leningrad/134/47/57 in ferrets.

The influenza A components of live attenuated vaccines used in Russia have been prepared as reassortants of the cold-adapted (ca) H2N2 viruses, A/Leningrad/134/17/57-ca (Len/17) and A/Leningrad/134/47/57-ca (Len/47), and virulent epidemic strains. The lesions responsible for attenuation within the six internal genes of each donor strain have been sequenced and described, but relatively little is known as to their stability before and after passage in susceptible hosts. In the work reported in this paper, RT-PCR restriction analysis and limited sequencing of individual genes were used to evaluate the stability of lesions in stocks of the both donor strains after passage in ferrets, which have been used widely as susceptible hosts for assessment of the virulence of influenza strains. Len/47 was shown to possess expected lesions by RT-PCR and restriction analysis. Substitution at position 1066 of the NP gene, which has been previously reported to be unique to Len/47 [Klimov et al., Virology 186 (1992) 795], was also shown to be present in all clones of Len/17. This change was confirmed by limited sequence analysis and was shown to be retained in progeny viruses isolated from the lungs and turbinates of inoculated ferrets. Two other changes in the PB2 and PB1 genes that were present in Len/47 were detected by limited sequence analysis alone. Further previously unreported minor changes were shown to be present for Len/17 and Len/47, but not both, and their significance is unknown. Limited replication of each donor strain occurred in ferrets and minimal clinical signs and histopathology were present. By contrast, the parental strain Len/57 and the recent epidemic strain A/Sydney/6/97 induced clinical signs and histopathology that were typical of influenza disease.

Adaptation, Physiological↗
PubMed1986

Biological characteristics of a cold-adapted influenza A virus mutation residing on a polymerase gene.

The biological function of a cold-adapted (ca) mutation residing on the PB2 gene of an influenza A/Ann Arbor/6/60 (A/AA/6/60) ca variant virus in the viral replication cycle at 25 degrees C was studied. The viral polypeptide synthesis of A/AA/6/60 ca variant at 25 degrees C was evident approximately 6 hours earlier than the wild type (wt) virus and yielded twice as many products. The quantitative analysis of viral complementary RNA (cRNA), synthesized in the presence of cycloheximide, revealed that A/AA/6/60 ca variant and a single gene reassortant that contains only the PB2 gene of the ca variant with remaining genes of the wt virus produced equal amount of cRNA at 25 degrees and 33 degrees C, which was an amount approximately four fold greater than the wt virus' cRNA synthesized at 25 degrees C. These results strongly suggest that the ca mutation residing on the PB2 gene of A/AA/6/60 ca variant affects the messenger RNA synthesis at 25 degrees C in the primary transcription.

Cold Temperature↗
PubMed1993

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.

Administration, Intranasal↗