4 Notes
1. pH must be adjusted to the correct pH of 7.4 before use.
2. Integrity and quality of RNA must be checked before further
downstream processes by either NanoDrop or Qubit or by
agarose gel imaging.
3. Control PCR must be done with DNase treated sample before
and after cDNA conversion for detection of successful reverse
transcription.
4. Mutations are required to be introduced at sites not involved in
packaging and splicing of viral RNP [53–55].
5. Incorporation of mutations must be at sites exhibiting higher
than 99% conservation at amino acid sequence but not at
nucleotide level in order to avoid the risk of adding mutations
at “mutational hot spots” or causing loss of conserved codon
positions serving as “potential critical RNA signals” [34–36].
6. Out-of-frame ORFs must be excluded from mutagenesis.
7. A major advantage of creating of genetically attenuated parasite
vaccine is that they exhibit identical genetic identity by existing
in a homogeneous population. However, attenuation by a
single crossover event can lead to reversion to wild type parasite. Thus, double crossover recombination can prevent such
problems of reversion [7].
References
1. Badgett MR, Auer A, Carmichael LE, Parrish
CR, Bull JJ (2002) Evolutionary dynamics of
viral
attenuation.
J
Virol
76
(20):10524–10529.
https://doi.org/10.
1128/jvi.76.20.10524-10529.2002
2. Ebert D (1998) Experimental evolution of
parasites. Science 282(5393):1432–1435.
https://doi.org/10.1126/science.282.5393.
1432
3. Dragunsky
EM,
Rivera
E,
Donald
Hochstein H, Levenbook IS (1990) In vitro
characterization of Salmonella typhi mutant
strains for live oral vaccines. Vaccine 8
(3):263–268.
https://doi.org/10.1016/
0264-410X(90)90056-R
4. Kirkpatrick BD, McKenzie R, O’Neill JP, Larsson CJ, Bourgeois AL, Shimko J, Bentley M,
Makin J, Chatfield S, Hindle Z, Fidler C,
Robinson BE, Ventrone CH, Bansal N, Carpenter CM, Kutzko D, Hamlet S, Lapointe C,
Taylor DN (2006) Evaluation of Salmonella
enterica serovar Typhi (Ty2 aroC-ssaV-)
M01ZH09, with a defined mutation in the
Salmonella pathogenicity island 2, as a live,
oral typhoid vaccine in human volunteers. Vaccine 24(2):116–123. https://doi.org/10.
1016/j.vaccine.2005.08.008
5. Hohmann EL, Oletta CA, Killeen KP, Miller SI
(1996) phoP/phoQ-deleted Salmonella typhi
(Ty800) is a safe and immunogenic single-dose
typhoid fever vaccine in volunteers. J Infect Dis
173(6):1408–1414.
https://doi.org/10.
1093/infdis/173.6.1408
6. Kaper JB, Michalski J, Ketley JM, Levine MM
(1994) Potential for reacquisition of cholera
enterotoxin genes by attenuated Vibrio cholerae vaccine strain CVD 103-HgR. Infect
Immun 62(4):1480–1483
7. Butler NS, Schmidt NW, Vaughan AM, Aly AS,
Kappe SHI, Harty JT (2011) Superior antimalarial immunity after vaccination with late liver
stage-arresting genetically attenuated parasites.
Cell Host Microbe 9(6):451–462. https://doi.
org/10.1016/j.chom.2011.05.008
8. Ravenel MP (1928) La Vaccination Pre ´ventive
Contre la Tuberculose par le “BCG.”. Am J
Live Vaccine Attenuation
353
1. pH must be adjusted to the correct pH of 7.4 before use.
2. Integrity and quality of RNA must be checked before further
downstream processes by either NanoDrop or Qubit or by
agarose gel imaging.
3. Control PCR must be done with DNase treated sample before
and after cDNA conversion for detection of successful reverse
transcription.
4. Mutations are required to be introduced at sites not involved in
packaging and splicing of viral RNP [53–55].
5. Incorporation of mutations must be at sites exhibiting higher
than 99% conservation at amino acid sequence but not at
nucleotide level in order to avoid the risk of adding mutations
at “mutational hot spots” or causing loss of conserved codon
positions serving as “potential critical RNA signals” [34–36].
6. Out-of-frame ORFs must be excluded from mutagenesis.
7. A major advantage of creating of genetically attenuated parasite
vaccine is that they exhibit identical genetic identity by existing
in a homogeneous population. However, attenuation by a
single crossover event can lead to reversion to wild type parasite. Thus, double crossover recombination can prevent such
problems of reversion [7].
References
1. Badgett MR, Auer A, Carmichael LE, Parrish
CR, Bull JJ (2002) Evolutionary dynamics of
viral
attenuation.
J
Virol
76
(20):10524–10529.
https://doi.org/10.
1128/jvi.76.20.10524-10529.2002
2. Ebert D (1998) Experimental evolution of
parasites. Science 282(5393):1432–1435.
https://doi.org/10.1126/science.282.5393.
1432
3. Dragunsky
EM,
Rivera
E,
Donald
Hochstein H, Levenbook IS (1990) In vitro
characterization of Salmonella typhi mutant
strains for live oral vaccines. Vaccine 8
(3):263–268.
https://doi.org/10.1016/
0264-410X(90)90056-R
4. Kirkpatrick BD, McKenzie R, O’Neill JP, Larsson CJ, Bourgeois AL, Shimko J, Bentley M,
Makin J, Chatfield S, Hindle Z, Fidler C,
Robinson BE, Ventrone CH, Bansal N, Carpenter CM, Kutzko D, Hamlet S, Lapointe C,
Taylor DN (2006) Evaluation of Salmonella
enterica serovar Typhi (Ty2 aroC-ssaV-)
M01ZH09, with a defined mutation in the
Salmonella pathogenicity island 2, as a live,
oral typhoid vaccine in human volunteers. Vaccine 24(2):116–123. https://doi.org/10.
1016/j.vaccine.2005.08.008
5. Hohmann EL, Oletta CA, Killeen KP, Miller SI
(1996) phoP/phoQ-deleted Salmonella typhi
(Ty800) is a safe and immunogenic single-dose
typhoid fever vaccine in volunteers. J Infect Dis
173(6):1408–1414.
https://doi.org/10.
1093/infdis/173.6.1408
6. Kaper JB, Michalski J, Ketley JM, Levine MM
(1994) Potential for reacquisition of cholera
enterotoxin genes by attenuated Vibrio cholerae vaccine strain CVD 103-HgR. Infect
Immun 62(4):1480–1483
7. Butler NS, Schmidt NW, Vaughan AM, Aly AS,
Kappe SHI, Harty JT (2011) Superior antimalarial immunity after vaccination with late liver
stage-arresting genetically attenuated parasites.
Cell Host Microbe 9(6):451–462. https://doi.
org/10.1016/j.chom.2011.05.008
8. Ravenel MP (1928) La Vaccination Pre ´ventive
Contre la Tuberculose par le “BCG.”. Am J
Live Vaccine Attenuation
353