Level Of Temperature Sensitivity Research Articles

A Mutated PB1 Residue 319 Synergizes with the PB2 N265S Mutation of the Live Attenuated Influenza Vaccine to Convey Temperature Sensitivity.

Current influenza vaccines have modest efficacy. This is especially true for current live attenuated influenza vaccines (LAIV), which have been inferior to the inactivated versions in recent years. Therefore, a new generation of live vaccines may be needed. We previously showed that a mutation at PB1 residue 319 confers enhanced temperature sensitivity and attenuation in an LAIV constructed in the genetic background of the mouse-adapted Influenza A Virus (IAV) strain A/PR/8/34 (PR8). Here, we describe the origin/discovery of this unique mutation and demonstrate that, when combined with the PB2 N265S mutation of LAIV, it conveys an even greater level of temperature sensitivity and attenuation on PR8 than the complete set of attenuating mutations from LAIV. Furthermore, we show that the combined PB1 L319Q and PB2 N265S mutations confer temperature sensitivity on IAV polymerase activity in two different genetic backgrounds, PR8 and A/Cal/04/09. Collectively, these findings show that the PB2 LAIV mutation synergizes with a mutation in PB1 and may have potential utility for improving LAIVs.

A novel strategy to improve the sensitivity of aerosol phosphor thermometry using co-doped phosphors

A novel strategy is presented to improve the temperature sensitivity and potentially extend the temperature range of aerosol phosphor thermometry (APT) by co-doping host materials with two rare-earth ions. The ratio of the emission bands from each ion are measured and calibrated versus temperature to utilize the high sensitivity of thermographic phosphor absolute signal levels. The potential of the technique is illustrated using trivalent cerium (Ce3+) and praseodymium (Pr3+) co-doped into yttrium aluminum garnet (Ce,Pr:YAG). The measured fractional sensitivity of this phosphor from 300–450 K was 0.004–0.006 K−1, a factor of 1.5–2 better than previously observed for Eu:BAM. Additionally, the single-shot precision (1σ) was between 9 and 25 K over the range of temperatures measured, illustrating the utility of this co-doping strategy. The level of temperature sensitivity and single-shot precision observed here should be achievable over different temperature ranges by doping Ce3+ and Pr3+ into different hosts. This new strategy should provide a pathway to ultimately extend the high-temperature single-shot measurement limit for APT to temperatures greater than 1000 K and push forward the state-of-the-art for planar temperature diagnostics in combustion applications.

The two amino acid substitutions in the L protein of cpts530/1009, a live-attenuated respiratory syncytial virus candidate vaccine, are independent temperature-sensitive and attenuation mutations

cpts530/1009 is a live-attenuated, temperature-sensitive ( ts) RSV vaccine candidate that was shown previously to be attenuated for seronegative humans. It was generated by two rounds of chemical mutagenesis: first, a partially attenuated, cold-passaged ( cp), non- ts RSV mutant ( cpRSV) was mutagenized to yield the ts derivative cpts530, and then cpts530 was mutagenized to yield cpts530/1009, which is more ts. Previous nucleotide (nt) sequence analysis of cpts530 showed that it has a single nt change compared to cpRSV that results in an amino acid substitution at residue 521 in the L protein. Reverse genetics confirmed that this mutation is responsible for the ts phenotype of cpts530. Here, determination of the complete 15,222-nt sequence of cpts530/1009 identified a single change compared to cpts530, namely a point mutation at nt 12002, which results in a methionine-to-valine substitution at amino acid 1169 in the L protein. The contribution of the 1009 mutation to the level of temperature sensitivity and attenuation exhibited by cpts530/1009 was evaluated by its introduction alone or with the 530 and cp mutations into the full-length cDNA clone of wild-type (wt) RSV. Subsequent analysis of infectious viruses recovered from the mutant cDNAs indicated that (i) the 1009 mutation indeed was a ts mutation and the level of temperature sensitivity specified by the 1009 mutation was less than that specified by the 530 mutation, (ii) the 530 and 1009 mutations each contributed to attenuation in the upper respiratory tract of mice and their effects were additive, (iii) viruses bearing the 1009 mutation were more attenuated in the lower respiratory tract of mice than viruses bearing the 530 mutation and (iv) the combination of the 530 and 1009 mutations in the cpRSV background resulted in the same level of temperature sensitivity and attenuation in mice as that observed for the biologically-derived cpts530/1009 mutant. These data show that the genetic basis of the attenuation and temperature sensitivity of the cpts530/1009 candidate vaccine virus is the sum of the contributions of seven identified amino acid substitutions, i.e. the 5 cpRSV mutations, the 530 mutation and the 1009 mutation.

Identification of mutations contributing to the temperature-sensitive, cold-adapted, and attenuation phenotypes of the live-attenuated cold-passage 45 (cp45) human parainfluenza virus 3 candidate vaccine.

The live-attenuated human parainfluenza virus 3 (PIV3) cold-passage 45 (cp45) candidate vaccine was shown previously to be safe, immunogenic, and phenotypically stable in seronegative human infants. Previous findings indicated that each of the three amino acid substitutions in the L polymerase protein of cp45 independently confers the temperature-sensitive (ts) and attenuation (att) phenotypes but not the cold-adaptation (ca) phenotype (29). cp45 contains 12 additional potentially important point mutations in other proteins (N, C, M, F, and hemagglutinin-neuraminidase [HN]) or in cis-acting sequences (the leader region and the transcription gene start [GS] signal of the N gene), and their contribution to these phenotypes was undefined. To further characterize the genetic basis for the ts, ca, and att phenotypes of this promising vaccine candidate, we constructed, using a reverse genetics system, a recombinant cp45 virus that contained all 15 cp45-specific mutations mentioned above, and found that it was essentially indistinguishable from the biologically derived cp45 on the basis of plaque size, level of temperature sensitivity, cold adaptation, level of replication in the upper and lower respiratory tract of hamsters, and ability to protect hamsters from subsequent wild-type PIV3 challenge. We then constructed recombinant viruses containing the cp45 mutations in individual proteins as well as several combinations of mutations. Analysis of these recombinant viruses revealed that multiple cp45 mutations distributed throughout the genome contribute to the ts, ca, and att phenotypes. In addition to the mutations in the L gene, at least one other mutation in the 3' N region (i.e., including the leader, N GS, and N coding changes) contributes to the ts phenotype. A recombinant virus containing all the cp45 mutations except those in L was more ts than cp45, illustrating the complex nature of this phenotype. The ca phenotype of cp45 also is a complex composite phenotype, reflecting contributions of at least three separate genetic elements, namely, mutations within the 3' N region, the L protein, and the C-M-F-HN region. The att phenotype is a composite of both ts and non-ts mutations. Attenuating ts mutations are located in the L protein, and non-ts attenuating mutations are located in the C and F proteins. The presence of multiple ts and non-ts attenuating mutations in cp45 likely contributes to the high level of attenuation and phenotypic stability of this promising vaccine candidate.

A Single Nucleotide Substitution in the Transcription Start Signal of the M2 Gene of Respiratory Syncytial Virus Vaccine Candidatecpts248/404 Is the Major Determinant of the Temperature-Sensitive and Attenuation Phenotypes

Respiratory syncytial virus (RSV)cpts248/404 is a live-attenuated, temperature-sensitive (ts) vaccine candidate derived from cold-passagedcpRSV by two rounds of chemical mutagenesis and biological selection. Previous sequence analysis showed that these two steps introduced three single nucleotide substitutions into thecpRSV parent. Two of these occurred within the coding sequence for the L protein, and each resulted in a single amino acid substitution: Gln-831-Leu (248 mutation) and Asp-1183-Glu (404-L mutation). The third mutation resulted in a nucleotide substitution at position 9 of thecis-acting gene start signal of the M2 gene (404-M2 mutation). In the present study, the genetic basis of attenuation ofcpts248/404 was defined by the introduction of each of these mutations (singly or in combination) into a full-length cDNA clone ofcpRSV. Recombinant RSV derived from each mutant cDNA was analyzed to determine the contribution of each mutation to thetsand attenuation phenotypes of the virus. This analysis showed that the 248 mutation specifies a significant reduction of plaque formation at 38°C and is responsible for an intermediate level of attenuation in mice. In contrast, the 404-L mutation did not contribute to thetsor attenuation phenotype alone or in combination with other mutations and is thus an incidental change. Unexpectedly, the 404-M2 mutation alone specified complete restriction of plaque formation at 37°C and a high level of attenuation in mice. This indicates that the level of temperature sensitivity and attenuation ofcpts248/404 can be attributed primarily to the 404-M2 mutation. Thus thecpts248/404 virus contains a set oftsand non-tsattenuating mutations, which likely accounts for its genetic stability. The recombinant version of this virus, rA2cp248/404, was phenotypically indistinguishable fromcpts248/404 and represents a background into which additional mutations can be introduced as needed to obtain the desired level of attenuation for successful immunization of the very young human infant.

The temperature-sensitive (ts) phenotype of a cold-passaged (cp) live attenuated respiratory syncytial virus vaccine candidate, designated cpts530, results from a single amino acid substitution in the L protein.

cpts530, a candidate live-virus vaccine, is an attenuated strain of human respiratory syncytial virus (RSV). It was derived by subjecting a cold-passaged (cp) strain of RSV to a single round of chemical mutagenesis. cpts530 is a temperature-sensitive (ts) mutant that is attenuated in mice and chimpanzees, and its ts phenotype exhibits a high level of stability during replication in both species. In the present study, the complete nucleotide sequence of cpts530 RSV was determined. The five mutations known to be present in the parent cpRSV were retained in its cpts530 derivative, and one additional nucleotide change was identified at nucleotide (nt) 10060, which resulted in a phenylalanine-to-leucine change at amino acid 521 in the large polymerase (L) protein. To determine if this single amino acid substitution was indeed responsible for the ts phenotype of cpts530, it was introduced alone or in combination with the cp mutations into the full-length cDNA clone of the wild-type A2 RSV. Analysis of infectious viruses recovered from mutant cDNAs indicated that this single mutation specified complete restriction of plaque formation of recombinant cp530 in HEp-2 cell monolayer cultures at 40 degrees C, and the level of temperature sensitivity was not influenced by the presence of the five cpRSV mutations. These findings identify the phenylalanine-to-leucine change at amino acid 521 in the L protein as the mutation that specifies the ts phenotype of cpts530. Furthermore, these findings illustrate the feasibility of using the cDNA-based recovery system to analyze and construct defined attenuated vaccine viruses.

An influenza A live attenuated reassortant virus possessing three temperature-sensitive mutations in the PB2 polymerase gene rapidly loses temperature sensitivity following replication in hamsters

The purpose of the present study was to produce an influenza A H2N2 donor virus from which an attenuating PB2 gene bearing three discrete temperature sensitive ( ts) mutations could be readily transferred to currently epidemic influenza A H1N1 and H3N2 viruses via genetic reassortment. An influenza A transfectant virus was first produced that contained site-directed ts mutations at amino acids 112, 265, and 556 in the PB2 gene of influenza A/AA/60 virus origin in a background of the other seven RNA segments from the influenza A/LA/87 (H3N2) virus. The A/LA/87 PB2 ts transfectant virus (clone 22B1) was mated with the A/AA/60 (H2N2) wild type virus, and six H2N2 ts reassortants were obtained. One reassortant virus, clone 25A1, possessed the triple ts PB2 gene in the context of all seven other genes of homologous A/AA/60 origin. Isolation of this reassortant permitted an examination of the contribution of the ts mutations present in a triple ts PB2 transfectant virus to its attenuation and phenotypic stability indenpendent from an effect of the A/AA/60-A/LA/87 gene constellation on attenuation. It was found that the A/AA/60 triple ts reassortant virus was less ts, less attenuated, and less phenotypically stable than the A/LA/87 triple ts transfectant virus from which it was derived. The A/AA/60 reassortant possessing the PB2 gene containing three introduced ts mutations underwent rapid and significant loss of its temperature sensitivity following replication in the lungs of immunocompetent hamsters. This indicated that the A/AA/60-A/LA/87 gene constellation contributed significantly to the overall level of temperature-sensitivity, attenuation, and stability of the A/LA/87 triple ts transfectant virus. It is likely that the instability of the ts phenotype exhibited by the A/AA/60 triple ts reassortant virus would not be acceptable for a vaccine to be used in humans. The implications of these findings for the usefulness of ts mutations as the sole attenuating mutation in influenza virus vaccines is discussed.

Nucleotide Sequence Analysis of the Respiratory Syncytial Virus Subgroup A Cold-Passaged (cp) Temperature Sensitive (ts)cpts-248/404 Live Attenuated Virus Vaccine Candidate

The complete nucleotide sequence of the RSVcpts-248/404 live attenuated vaccine candidate was determined from cloned cDNA and was compared to that of the RSV A2/HEK7 wild-type, cold-passagedcp-RSV, andcpts-248 virus, which constitute the series of progenitor viruses. RSVcpts-248/404 is more attenuated and more temperature sensitive (ts) (shut-off temperature 36°) than itscpts-248 parent virus (shut-off temperature 38°) and is currently being evaluated in phase I clinical trials in humans. Our ultimate goal is to identify the genetic basis for the host range attenuation phenotype exhibited bycp-RSV (i.e., efficient replication in tissue culture but decreased replication in chimpanzees and humans) and for thetsand attenuation phenotypes of its chemically mutagenized derivatives,cpts-248 andcpts-248/404. Compared with itscpts-248 parent, thecpts-248/404 virus possesses an amino acid change in the polymerase (L) protein and a single nucleotide substitution in the M2 gene start sequence. In total, thecpts-248/404 mutant differs from its wild-type RSV A2/HEK7 progenitor in seven amino acids [four in the polymerase (L) protein, two in the fusion (F) glycoprotein, and one in the (N) nucleoprotein] and one nucleotide difference in the M2 gene start sequence. Heterogeneity at nucleotide position 4 (G or C, negative sense, compared to G in the RSV A2/HEK7 progenitor) in the leader region of vRNA developed during passage of thecpts-248/404 in tissue culture. Biologically cloned derivatives of RSVcpts-248/404 virus that differed at position 4 possessed the same level of temperature sensitivity and exhibited the same level of replication in the upper and lower respiratory tract of mice, suggesting that heterogeneity at this position is not clinically relevant. The determination of the nucleotide sequence of thecpts-248/404 virus will allow evaluation of the stability of the eight mutations that are associated with the attenuation phenotype during vaccine production and following replication in humans.

Genetic factors associated with loss of the temperature-sensitive phenotype of the influenza A/Alaska/77-ts-1A2 recombinant during growth in vivo

The influenza A/Alaska/77- ts-1A2 vaccine candidate strain, which has a 37° shutoff temperature for plaque formation and temperature-sensitive ( ts) lesions on the P1 and P3 genes, was administered intranasally to a seronegative child who subsequently remained well. However, starting 7 days after inoculation, A/Alaska/77 virus with a 40° shutoff temperature ( ts +) was shed. To determine whether an extragenic suppressor mutation was responsible for this loss of ts phenotype of the vaccine candidate virus, the ts + isolate was mated with wild-type virus, and ts recombinant progeny were sought. Approximately 25% of the progeny were ts, suggesting that a suppressor mutation was present in the child's ts + isolate. Each of 62 ts progeny viruses had the ts P3 gene. The extragenic suppressor of this ts gene was localized to the P2 gene. The ts P3 gene of the A/Alaska/77- ts-1A2 recombinant specified a 37–38° shutoff temperature for plaque formation, whereas the ts P3 gene in the ts segregants derived from the ts + isolate had a 39° shutoff temperature. Hence, the ts P3 gene in the ts + isolate developed an intragenic mutation affecting its level of temperature sensitivity. In addition, the P3 RNA of the ts + virus migrated more slowly in polyacrylamide gel than the P3 RNA of its A/Alaska/77- ts-1A2 parent. We failed to isolate ts P1 segregants from the ts + isolate, indicating that the P1 ts mutation had reverted or undergone intragenic suppression. The ability of ts mutants to escape from their ts phenotype by suppression and other mutations may limit their usefulness in immunoprophylaxis since attenuation of ts mutants of influenza virus appears to be a function of the ts phenotype. Additionally, suppressor mutations provide new opportunities for studying the nature of protein-protein interactions.