Codon optimization of Iranian human papillomavirus ... - Future Medicine

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Codon optimization of Iranian human papillomavirus Type 16 E6 oncogene for Lactococcus lactis subsp. cremoris MG1363 Sedigheh Taghinezhad-S1 , Vadood Razavilar*,2 , Hossein Keyvani3 , Mohammad Reza Razavi4 & Taher Nejadsattari5 1

Department of Microbiology, Faculty of Basic Sciences, Science & Research Branch, Islamic Azad University, Tehran, Iran Department of Food Hygiene, Faculty of Veterinary Sciences, Science & Research Branch, Islamic Azad University, Tehran, Iran 3 Department of Virology, Faculty of Medicine, Iran University of Medical Sciences, Tehran, IR Iran 4 Department of Parasitology, Pasteur Institute of Iran, Tehran, IR Iran 5 Department of Biology, Faculty of Basic Sciences, Science & Research Branch, Islamic Azad University, Tehran, IR Iran *Author for correspondence: Tel.: +98 21 44865179 82, +98 912 1026502; Fax: +98 21 44865179 82; [email protected] 2

Aim: The present study aimed to investigate the effect of codon optimization on E6 recombinant protein production in Lactococcus lactis. Method: Here we define the construction of shuttle vector harboring wild-type and codon-optimized HPV16 E6 oncogene, with maximum number of infrequent codons exchanged with codons that are frequently used in Lactococcus lactis subsp. cremoris MG1363. Results: Hence, the codons encoding 159 amino acids were modified, in which a total of 91 codons were changed, resulting in approximately threefold increase in protein expression of recombinant E6 (rE6). Conclusion: Our data revealed that codon usage optimization according to L. lactis desired codon usage can dramatically increase the expression of HPV16 E6, suggesting that this strategy is a valuable approach for immunization through DNA vaccine. First draft submitted: 8 March 2017; Accepted for publication: 5 June 2017; Published online: 29 September 2017 Keywords • E6 • HPV16 • human papillomaviruses • Lactococcus lactis

Background Cervical cancer is the second leading cause for cancer-related deaths among women worldwide. Human papillomaviruses (HPV), particularly HPV16, are associated with most cervical cancers [1]. The E6 and E7 proteins, which are usually expressed in all cervical cancers, are able to disrupt cell-cycle control by inactivating the tumor suppressors p53 and pRB via the ubiquitin-dependent proteolytic pathway [2,3]. Hence, HPV16 E6 and E7 are appropriate target antigens for developing vaccines. Of these two HPV16 oncogenes, E6 has been found to display additional variations than E7, which is moderately conserved. These sequence polymorphisms have been recognized in different geographical locations and ethnicity of each population [4,5]. The geographical relatedness of the virus has resulted in further taxonomy of HPV16 into five distinct molecular variants identified as the HPV16 European, African type II, African type I, Asian American and Asian variants [6,7]. HPV16 E6 oncogene is one of the best candidates for therapeutic HPV vaccines against cervical carcinoma. In this perspective, several outcomes have examined the use of genetically improved Gram-positive lactic acid bacteria (LAB) as E6 antigen delivery system to stimulate an immune response against HPV16. LAB are food-grade bacteria with a Generally Regarded As Safe status. They can be used for the delivery of recombinant proteins in foodstuff or in the digestive tract [8]. In the last two decades, genetic tools for the model LAB, Lactococcus lactis, were established [9,10]. Furthermore L. lactis genome is completely sequenced [11]. Many protein expression and targeting systems have also been designed for L. lactis [12]. These systems have been used to engineer L. lactis for the intracellular or extracellular creation of many viral, bacterial and eukaryotic proteins [13].

C 2017 Future Medicine Ltd 10.2217/fvl-2017-0032

Future Virol. (Epub ahead of print)

ISSN 1746-0794

Research Article

Taghinezhad-S, Razavilar, Keyvani, Razavi & Nejadsattari

Table 1. Bacterial strains, plasmids and primer used in this study. Strain/plasmid/primer

Relevant characteristics

Source

Host for pTZ57R/T vectors recA+, nalidixic acid resistant

Invitrogen

E. coli MC1061

recA+ for replication of pNZ8148

MoBiTec GmbH

Lactococcus lactis subsp. cremoris strain NZ9000

nisR and nisK integrated into peptidase pepN MG1363 MoBiTec GmbH pepN::nisRK. Most commonly used host of the NICE system, plasmid free

Strain Escherichia coli JM107

Plasmid pTZ57R/T

TA cloning vector, ampicillin resistant

Thermo scientific

pNZ8148

NICE system expression plasmid. Cmr, carries the nisin-inducible promoter PnisA

MoBiTec GmbH

HPV16-E6-F

CCATGGCACCAAAAGAGAACTGCAATG

This work

HPV16-E6-R

GAGCTCCAGCTGGGTTTCTCTACGTGTTC

This work

HPV16E6-Opti-F

CATCAAAAACGTACTGCTATGTTTC

This work

HPV16E6-Opti-R

GAGTTTCACGACGAGTACGTGATG

This work

pNZ8148-F

GATAACGCGAGCATAATAAACGGC

This work

pNZ8148-R

GTTCTATCGAAAGCGAAATCAAACG

This work

Primer

Previous studies have reported that an effective approach for improving the expression levels of heterologous genes is codon optimization [14]. It has been described in bacteria [15], yeast [16], plants [17], mammalian cells [18] and filamentous fungi [19]. In the present study, we aimed to evaluate the nucleotide variations of the HPV16 E6 isolates in order to classify the different genetic variants publicized among infected women with cervical carcinoma in Iran. Also, we attempted to develop a DNA plasmid encoding optimized codon HPV16 E6 gene for L. lactis subsp. cremoris MG1363. Materials & methods Strains & plasmids Lactococcus lactis NZ9000, derived from L. lactis subsp. cremoris MG1363 and plasmid pNZ8148 (MoBiTec, Goettingen, Germany), was generously provided by H Keyvani from Iran University of Medical Sciences, Tehran, Iran. E. coli MC1061 was purchased from MoBiTec Corp (MoBiTec; Table 1). Study population

A total of 166 tumor specimens from Iranian women aged 18–62 years with a history of cervical cancer were included in the present study. Patients from our screening study were referred to Keyvan Virology Specialty Laboratory between May 2015 and April 2016. Informed consent for the study was granted. Tissue samples from lesions were achieved during the surgical procedure. Cervical cancer cells were embedded in paraffin blocks. HPV DNA detection

After deparaffinization of samples in xylene and rehydration in ethanol, DNA was extracted from the tissues using QIAmp Tissue Kit (Qiagen GmbH, Hilden, Germany) following the manufacturer’s protocol. All samples were selected for the PCR amplification with primers MY09/MY11 placed within the conserved regions of HPV L1 gene. To determine that all samples originally contained DNA of enough quality and quantity, samples were co-amplified for the occurrence of an internal standard, in this situation, β-globin [20]. INNO-LiPA assay

The INNO-LiPA HPV genotyping technique is based on amplification of a wide range of HPV genotypes with biotinylated SPF10 primers, targeting a 65-bp segment in the viral L1 region [21]. PCR was accomplished according to the manufacturer’s instructions. Briefly, 10 μl of PCR biotinylated products were denaturated and hybridized with type-specific oligonucleotide probes immobilized as parallel lines on nitrocellulose membrane strips. The hybrids were identified with alkaline phosphatase–streptavidin conjugate and substrates (5-bromo-4-chloro-3indolylphosphate and nitroblue tetrazolium), resulting in a purple precipitate at positive probe lines. After drying,

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Codon optimization of Iranian human papillomavirus Type 16 E6 oncogene for Lactococcus lactis subsp. cremoris MG1363

Research Article

Table 1. Bacterial strains, plasmids and primer used in this study. Strain/plasmid/primer

Relevant characteristics

Source

Host for pTZ57R/T vectors recA+, nalidixic acid resistant

Invitrogen

E. coli MC1061

recA+ for replication of pNZ8148

MoBiTec GmbH

Lactococcus lactis subsp. cremoris strain NZ9000

nisR and nisK integrated into peptidase pepN MG1363 MoBiTec GmbH pepN::nisRK. Most commonly used host of the NICE system, plasmid free

Strain Escherichia coli JM107

Plasmid pTZ57R/T

TA cloning vector, ampicillin resistant

Thermo scientific

pNZ8148

NICE system expression plasmid. Cmr, carries the nisin-inducible promoter PnisA

MoBiTec GmbH

HPV16-E6-F

CCATGGCACCAAAAGAGAACTGCAATG

This work

HPV16-E6-R

GAGCTCCAGCTGGGTTTCTCTACGTGTTC

This work

HPV16E6-Opti-F

CATCAAAAACGTACTGCTATGTTTC

This work

HPV16E6-Opti-R

GAGTTTCACGACGAGTACGTGATG

This work

pNZ8148-F

GATAACGCGAGCATAATAAACGGC

This work

pNZ8148-R

GTTCTATCGAAAGCGAAATCAAACG

This work

Primer

the strips were examined visually from an interpretation grid provided in the kit (Fujirebio, Gent, Belgium); the existence of a clearly visible line was considered to be a positive reaction. A biotinylated poly(dT) control for conjugate reaction was applied to each strip to warrant good efficiency of the test and appropriate alignment of the strips on the interpretation sheet. Evaluation of Iranian HPV16 E6 gene

Open reading frame (ORF) of HPV16 E6 gene was amplified through PCR using primers HPV16-E6-F and HPV16-E6-R (Table 1). Underlines illustrate the NcoI and SacI sites, respectively. The PCR conditions were as follows: 5 min at 94◦ C; 15 s at 94◦ C, 50 s at 55◦ C, 50 s at 72◦ C for 40 cycles; and a final extension of 15 min at 72◦ C. The PCR products of HPV16 E6 (483 bp), were purified by QIAquick PCR purification kit (Qiagen, Hilden, Germany). The pure PCR products were connected to pTZ57R/T vector (InsTAcloneTM PCR Cloning Kit; Thermo Fisher Scientific, Waltham, USA), and then the recombinant plasmid (which were named as pTZ57R/T-HPV16-E6) was transformed into E. coli JM107 competent cells. The recombinant plasmid was then extracted and submitted for sequencing via the universal M13 primer (Bioneer, Korea). Multiple sequence alignment performed using the CLC sequence viewer software (CLC bio, MA, USA). Accordingly, nucleotide sequences of the complete E6 ORF (477 bp/159 aa) in all 79 cancer patients screened were compared with the HPV16 reference sequence (ACCESSION: NC 001526). Codon bias & optimization

There are numerous methods to quantify the codon bias in a gene. The most frequently used and favored method is by calculating the effective number of codons in a gene. This is a number between 20 and 61 [22,23]. The codon adaptation index (CAI) value appears to be a valuable tool for appraising the expression level of heterologous genes. The CAI value is a number between 0 and 1 and describes to what degree the set of codons in a gene matches the set of codons in a reference set of differentially expressed genes [24]. Hence, the coding sequences of Iranian-derived sequences of E6 gene (in FASTA format) having correct codons with an exact multiple of three bases were subjected to codon usage analysis. For this purpose, the HPV16 E6 codon usage was optimized based on L. lactis codon usage [25]. We evaluated our analysis using the online optimizer server [26]. The corresponding gene sequences were constructed by Biomatik Company (Biomatik Corporation, Cambridge, Canada). Shuttle vector construction

The optimized E6 region from nt 7125–7601 of HPV16, was constructed in the backbone of a PMD18 vector containing NcoI and SacI sites at the 5 and 3 end, respectively. The resulted construct, pTZ57R/T-HPV16-E6 and pNZ8148 (MoBiTec) shuttle vector were digested using NcoI and SacI enzymes (New England Biolabs,


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70

60

50

30

Frequency: per thousand

40

20

10

0 GGAGGT AGAAGT CGA CGTTGCGAGGACAAG AAC CAG CAC TACGCGGCCACGACC CCGCCC TCG TCCGTG GTC ATA ATT CTA CTT TTA TTT G

R

S

R

W C

E

D

K

N

Q

H

Y

A

T

P

S

V

I

L

F

Codon of Lactococcus lactis subsp. cremoris MG1363

Figure 1.

Overall codon usage data of Lactococcus lactis subsp. cremoris MG1363 genes.

Table 2. Sequence alteration analysis of HPV16 E6 open reading frames in Iranian cervical samples. Position

Wild-type codon

Wild-type amino acid

Mutant codon

Mutant amino acid

Rate

49–51

AGA

Arginine (R)

ATA

Isoleucine (I)

(9/79) 11.39%

61–63

CAG

Glutamine (Q)

CAA

Glutamine (Q)

(38/79) 48.10%

94–96

GAT

Aspartic acid (D)

GAG

Glutamic acid (E)

(37/79) 46.83%

202–204

GCT

Alanine (A)

GGA

Glycine (G)

(6/79) 7.59%

211–213

GAT

Aspartic acid (D)

GAG

Glutamic acid (E)

(73/79) 92.40%

253–255

CAT

Histidine (H)

TAT

Tyrosine (Y)

(68/79) 86.07%

268–270

TTG

Leucine (L)

GTG

Valine (V)

(41/79) 51.89%

358–360

GAA

Glutamic acid (E)

GAC

Aspartic acid (D)

(58/79) 73.41 %

Ipswich, MA, USA). Digested HPV16-optiE6 and E6 sequences were inserted between the NcoI and SacI sites of the pNZ8148 plasmid to construct the pNZ8148-HPV16-optiE6 and pNZ8148-HPV16-E6 vector following manufacturer’s suggested conditions (MoBiTec). pNZ8148-HPV16-optiE6 and pNZ8148-HPV16-E6 plasmids were added to chemically competent E. coli MC1061 cells (MoBiTec). Bacteria were cultured on Luria–Bertani plates containing 10 μg/ml chloramphenicol. Plasmids having the correct insert were detected by colony PCR. The resulting shuttle vectors were approved by DNA sequencing. The pNZ8148-HPV16-E6 and pNZ8148-HPV16-optiE6 vectors were introduced into Electrocompetent L. lactis NZ9000 by electroporation using a Gene Pulser apparatus (Bio-Rad Laboratories, Inc., CA, USA) following the manufacturer’s instructions. Recombinant strains were screened on GM17 agar supplemented with 10 μg/ml chloramphenicol. The expression pNZ8148 vector without insert was electrotransformed into the L. lactis competent cells (negative control).

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Table 3. Codon usage table for the wild-type and codon-optimized Iranian E6 gene of HPV16 according to the relative codon usage frequencies of Lactococcus lactis subsp. cremoris MG1363. Amino acid

Codon

Host fraction

Wild-type HPV16 E6 number

Optimized HPV16 E6 number

F

TTT

0.76

4

5

TTC

0.24

1

0

TTA

0.31

8

0

TTG

0.22

2

0

CTT

0.26

0

15

CTC

0.08

0

0

CTA

0.08

0

0

CTG

0.06

5

0

ATT

0.68

3

9

ATC

0.21

0

0

ATA

0.11

6

0

GTT

0.48

0

5

GTC

0.19

0

0

GTA

0.2

3

0

GTG

0.14

2

0

L

I

V

S

P

T

A

Y

H

Q

N

K

D

E

C

W


TCT

0.25

2

0

TCC

0.05

0

0

TCA

0.33

2

6

TCG

0.06

0

0

CCT

0.36

1

0

CCC

0.09

1

0

CCA

0.46

4

7

CCG

0.09

1

0

ACT

0.36

2

9

ACC

0.13

2

0

ACA

0.39

5

0

ACG

0.12

0

0

GCT

0.41

1

2

GCC

0.17

0

0

GCA

0.31

1

0

GCG

0.11

0

0

TAT

0.78

9

11

TAC

0.22

2

0

CAT

0.75

3

4

CAC

0.25

1

0

CAA

0.84

8

13

CAG

0.16

5

0

AAT

0.79

2

4

AAC

0.21

2

0

AAA

0.83

4

11

AAG

0.17

7

0

GAT

0.72

3

7

GAC

0.28

4

0

GAA

0.82

4

10

GAG

0.18

6

0

TGT

0.76

10

14

TGC

0.24

4

0

TGG

1

1

1

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1.05 1.00 0.95 0.90 0.85 0.80 0.75

Relative adaptiveness (Wij)

0.70 0.65 0.60 0.55 0.50 0.45 0.40 0.35 0.30 0.25 0.20 0.15 0.10 0.05 0.00 10

20

30

40

50

60

70

80

90

100

110

120

130

140

150

Codons Relative adaptiveness (Wij)

Mean codon usage

Figure 2. The plot showing correlation between relative addictiveness and wild-type HPV16 E6 codons in Lactococcus lactis subsp. cremoris MG1363.

Table 3. Codon usage table for the wild-type and codon-optimized Iranian E6 gene of HPV16 according to the relative codon usage frequencies of Lactococcus lactis subsp. cremoris MG1363 (cont.). Amino acid

Codon

Host fraction

Wild-type HPV16 E6 number

Optimized HPV16 E6 number

R

CGT

0.4

2

17

CGC

0.12

0

0

CGA

0.15

3

0

CGG

0.06

2

0

AGT

0.22

2

0

AGC

0.09

0

0

R

AGA

0.22

8

0

AGG

0.04

2

0

G

GGT

0.37

2

5

GGC

0.13

0

0

GGA

0.38

2

0

GGG

0.13

1

0

S

Expression of recombinant E6 (rE6) in L. lactis

The recombinant L. lactis having shuttle vector pNZ8148-HPV16-E6, pNZ8148-HPV16-optiE6, and pNZ8148 were grown overnight in 50 ml GM17 supplemented with 10 μg/ml chloramphenicol at 30◦ C without aeration.

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M Query Optimized

H ATG CAC | | | | |# ATG CAT

Q K CAA AAG | | | | |# CAA AAA

R T A M F Q D AGA ACT GCA ATG TTT CAG GAC * | * | | | | | * | | | | | | | |# | | # CGT ACT GCT ATG TTT CAA GAT

P Q E CCA CAG GAG | | | | | # | |# CCA CAA GAA

R P I K L P CGA CCC ATA AAG TTA CCA | | * | | * | | * | |# # | * | | | CGT CCA ATT AAA CTT CCA

Query

I L E C V Y C L Q T T I H E I L C T E CAA TTA TGC ACA GAG CTG CAA ACA ACT ATA CAT GAG ATA ATA TTA GAA TGT GTG TAC TGC | | | #| * | |# | | * | |# | | * | | | | | * | | | | | * | | | | |# | | * | | * #| * | | | | | | | | * | |# | |#

Optimized

CAA CTT TGT ACT GAA CTT CAA ACT ACT ATT CAT GAA ATT ATT CTT GAA TGT GTT TAT TGT

Q

K Query Optimized

Q

Q

L

L

R

R

E

V

Y

D

F

A

F

R

D

L

C

I

V

AAG CAA CAG TTA CTG CGA CGT GAG GTA TAT GAC TTT GCT TTT CGG GAT TTA TGC ATA GTA | |# | | | | |# #| * | | * | | * | | | | |# | | * | | | | |# | | | | | | | | | | | * | | | #| * | |# | | * | | * AAA CAA CAA CTT CTT CGT CGT GAA GTT TAT GAT TTT GCT TTT CGT GAT CTT TGT ATT GTT Y

R

D

G

N

P

Y

G

V

C

E

K

C

L

K

F

Y

S

K

I

Query

TAT AGA GAT GGG AAT CCA TAT GGA GTA TGT GAG AAA TGT TTA AAG TTT TAT TCT AAA ATT

Optimized

| | | * | * | | | | | * | | | | | | | | | | | * | | * | | | | |# | | | | | | #| * | |# | | | | | | | | * | | | | | | TAT CGT GAT GGT AAT CCA TAT GGT GTT TGT GAA AAA TGT CTT AAA TTT TAT TCA AAA ATT S

E

Y

R

Y

Y

C

Y

S

V

Y

G

T

T

L

E

Q

Q

Y

N

Query

AGT GAG TAT AGA TAT TAT TGT TAT AGT GTG TAT GGA ACA ACA TTA GAA CAG CAA TAC AAC * ** | |# | | | * | * | | | | | | | | | | | | *** | | * | | | | | * | | * | | * #| * | | | | |# | | | | |# | |#

Optimized

TCA GAA TAT CGT TAT TAT TGT TAT TCA GTT TAT GGT ACT ACT CTT GAA CAA CAA TAT AAT K

Query Optimized

Query Optimized

Query Optimized

P

L

C

D

L

L

I

R

C

I

N

C

Q

K

P

L

C

P

D

AAA CCG TTG TGT GAT TTG TTA ATT AGG TGT ATT AAC TGT CAA AAG CCA CTG TGT CCT GAC | | | | |# #| * | | | | | | #| * #| * | | | * | * | | | | | | | |# | | | | | | | |# | | | | | * | | | | | * | |# AAA CCA CTT TGT GAT CTT CTT ATT CGT TGT ATT AAT TGT CAA AAA CCA CTT TGT CCA GAT Q K E GAA AAG CAA | | | | |# | | | GAA AAA CAA

R

C R G GGT CGA TGT | | | | |* | | | GGT CGT TGT

M

H

L

D

K

K

Q

R

F

H

N

I

R

G

R

W

T

AGA CAT CTG GAC AAA AAG CAA AGA TTC CAT AAT ATA AGG GGT CGG TGG ACC *|* | | | | |* | |# | | | | |# | | | *|* | |# | | | | | | | |* *|* | | | | |* | | | | |# CGT CAT CTT GAT AAA AAA CAA CGT TTT CAT AAT ATT CGT GGT CGT TGG ACT S

C

C

R

S

S

R

T

R

R

E

T

Q

L

.

ATG TCT TGT TGC AGA TCA TCA AGA ACA CGT AGA GAA ACC CAG CTG TAA | | | | |* | | | | |# *|* | | | | | | *|* | |* | | | *|* | | | | |# | |# | |* | | | ATG TCA TGT TGT CGT TCA TCA CGT ACT CGT CGT GAA ACT CAA CTT TAA

Figure 3. Alignment of nucleotide sequence between the optimized and wild-type E6 gene along with amino acid composition. —: Unchanged nucleotide; *: Transversion change (Purines Pyrimidines); #: Transition change (Purine Purine/Pyrimidine Pyrimidine).

Cultures at an OD600 = 0.5 were induced with 10 ng/ml of nisin (Sigma-Aldrich, Steinheim, Germany), and incubated for 4 h. Analysis of rE6 proteins

Cytoplasmic protein fractions were isolated separately, and loaded onto 12% acrylamide gels. SDS-PAGE and western blotting were carried out essentially as described in the standard procedure. Immunodetection of rE6 was carried out by the use of monoclonal Anti-HPV16 E6 + HPV18 E6 antibody as a primary antibody (ab7;0 Abcam Inc., Toronto, ON, Canada; 1:5000 dilution). The membrane was washed with TBST (Tris-buffered saline, 0.1%


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3000 bp

1500 bp

1000 bp

Figure 4. The double digestion patterns of the pNZ8148-HPV16-optiE6 shuttle vector via NcoI and SacI restriction endonuclease. Lane A: 100-bp ladder; Lane B: Digested pNZ8148-HPV16-optiE6 using NcoI and SacI enzyme; Lane C: Undigested pNZ8148-HPV16-optiE6.

500 bp 400 bp

Tween 20; 20 mM Tris–HCl, 150 mM NaCl, 0.1% Tween 20, pH 7.5), and incubated with Goat Anti-Mouse IgG H&L (HRP) antibody (ab6789; Abcam, Canada; 1:10,000 dilution) at room temperature for 1 h. Detection was carried out by the use HRP/3,3-diaminobenzidine (DAB) substrate. Protein bands were quantified by densitometry using the ImageJ software [27].

Results HPV detection & typing

Out of 166 samples, 163 (98.19%) cases were positive for HPV infection, whereas 1.80% (3/166) of cervical cancer biopsies were found to be HPV negative. On the basis of the results, distribution of the most common high-risk type single and multiple infections in the Iranian HPV-positive woman was HPV16 (79/163, 48.46%), singly or in combination, followed by HPV31 (16/163, 9.81%), HPV39 (13/163, 7.97%), HPV51 (11/163, 6.74%), HPV53 (10/163, 6.13%), HPV66, and HPV18 (9/163, 5.52% each), HPV58 (5/163, 3.06%), HPV35 (4/163, 2.45%), HPV52 (3/163, 1.84%), HPV56 (2/163, 1.22%), HPV45, and HPV68 (1/163, 0.61% each). There were 87/163 single infections (53.37%) and 26/163 multiple infections (46.62%) from the 166 HPV-positive patients. Among multiple infections, HPV31, HPV53, and HPV66 followed by HPV16, were the most commonly detected types.

Sequence analysis of HPV16 E6 oncogene

The nucleotide sequence of HPV16 E6 gene obtained from Iranian patient compared with HPV16 reference showed that all of the analyzed sequences contained at least one nucleotide modification in the E6 region. The nucleotide variation rate of HPV16 E6 was 100% (79 of 79) whereas the amino acid variation rate was 93.67% (74 of 79). The most frequently observed variations were nucleotide substitution at positions 213, 253, 268, and 360 which induced the amino acid replacement D71E, H85Y, L90V, and E120D, respectively. But the greatest observed variant was T213G. This led to an amino acid change of D71E in 92.40% of the samples. The common variant discovered from cervical samples was G63A (48.10%), but it did not lead to any amino acid (glutamine) change. We discovered a new E6 nucleotide mutation G50T (R17I) among the 11.39% (9/79) HPV16-positive patients. All nucleotide changes, variants and their prevalence of mutant codon along with amino acid in the ORF of Iranian HPV16 E6 are summarized in Table 2.

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53 KDa

41 KDa

30 KDa

22 KDa

Optimized-E6

Wild type-E6

Figure 5. Comparison of wild-type E6 and optiE6 for expression in Lactococcus lactis cells by SDS-PAGE. Lane A: Molecular weight marker; Lane B: Cytoplasmic fraction protein extract from nisin-induced Lactococcus lactis NZ9000 (pNZ8148-HPV16-optiE6); Lane C: Cytoplasmic fraction protein extract from nisin-induced Lactococcus lactis NZ9000 (pNZ8148-HPV16-E6).

Adaption of the E6 oncogene to the codon bias of L. lactis subsp. cremoris MG1363

Figure 1 showed the distribution of codon usage frequency presented in L. lactis subsp. cremoris MG1363. It was found via online analysis for DNA sequence of the native E6 from HPV16 that some AA residues were encoded by codons that are infrequently represented in L. lactis subsp. cremoris MG1363 (Figure 2), they are TTC (F), TTA/TTG/CTG (L), ATA (I), GTA/GTG (V), TCT/AGT (S), CCT/CCC/CCG (P), ACC/ACA (T), GCA (A), TAC (Y), CAC (H), CAG (Q), AAC (N), AAG (K), GAC (D), GAG (E), TGC (C), CGA/CGG/AGA/AGG (R), and GGA/GGG (G). Consequently, individual codons of the native E6 gene were investigated for the unfavorable presence of codon and were consecutively replaced using a similar triplet which substituted. Those codons best appropriate to the L. lactis cremoris subsp. cremoris MG1363 codon bias were favorably selected. Totally, the codons encoding 159 amino acids were optimized in which a total of 91 codons were improved. The optimized codons for each AA are summarized in Table 3. The modifications adjusted the GC and AT content of the entire optimized E6 gene to 31.40 and 68.60% overall. After optimization, the codon usage is very well adapted to the preferences in L. lactis cremoris subsp. cremoris MG1363 as verified by a CAI of 1.000 instead of 0.264. Furthermore, the effective number of codon values for optimized E6 oncogene was decreased from 49 to 21. Alignment results among optimized and native HPV16 E6 genes are presented in Figure 3. Construction of the shuttle vector

Based on the nucleotide sequence, the full optimized ORF of HPV16 E6 was cloned as described in the ‘Materials & methods’ section. Through digestion with NcoI and SacI restriction enzyme, pNZ8148-HPV16-optiE6 and pNZ8148-HPV16-E6 shuttle vectors were confirmed to be successfully created (Figure 4). The existence of the desired optiE6 and E6 genes in the L. lactis NZ9000 strains were further confirmed by digestion, PCR, and sequencing (data not shown). Analysis of rE6 oncoprotein

To examine the influence of codon optimization on protein expression levels, recombinant L. lactis was used to express oncoprotein E6. A strong intracellular signal (∼22 kDa) corresponding to the nisin-induced L. lactis harboring pNZ8148-HPV16-optiE6 and a weak band (∼22 kDa) representing the intracellular form of rE6 were detected on SDS-PAGE analysis of the cytoplasm extract of nisin-induced L. lactis NZ9000 harboring pNZ8148HPV16-E6 (Figure 5). As can be seen from Figure 5, the rE6 expression was about approximately threefold higher


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53 KDa

41 KDa

30 KDa

Wild type-E6 Optimized-E6

22 KDa

Figure 6. Western blot analysis of wild-type E6 and optiE6 for expression in Lactococcus lactis cells. Lane A: Molecular weight marker; Lane B: Cytoplasmic fraction protein extract from nisin-induced Lactococcus lactis NZ9000 (pNZ8148-HPV16-E6); Lane C: Cytoplasmic fraction protein extract from nisin-induced Lactococcus lactis NZ9000 (pNZ8148-HPV16-optiE6).

for the optiE6 than E6. The results showed that an empty vector pNZ8148 (negative control) and in the absence of inducer, recombinant proteins could not be detected in the recombinant NZ9000 strains. Western blot analysis revealed a single clear band in the nisin-induced cytoplasm fraction of both recombinant strain corresponding to the expected size (∼22 KDa) of rE6, and no signal was observed in noninduced NZ9000 cultures by western blotting (Figure 6). Discussion The present study is the first to demonstrate the efficiency of paraffin-embedded tissue for sequence examination of HPV16 E6 gene and codon optimization. In our study, the detection rate of HPV DNA (98.19%) was comparable to the worldwide prevalence (99.7%), but was larger than those from the earlier studies in the same country. The prevalence of multiple infections in our study varied from other studies of Iran (46.62%) [28–30]. These variations may be because of differences in geographic and ethnic characteristics, sexual behavior and genotyping methods. Our data are in agreement with previous studies of Iran representing genotypes 18, 31, 39, 52, 53, and 66 which were observed more frequently and elicited after HPV16 in Iranian HPV-positive women with cervical cancer [28,30,31]. A more recent study on HPV-type distribution in invasive cervical cancer confirmed these results, showing that the amount of HPV16 and HPV18 in Asia, Africa, and south/central America is minor than in Europe, north America, and Oceania while the incidence of HPV52 and HPV58 is relatively high in Asia [29,32]. Examination of the E6 nucleotide revealed a high degree of sequence heterogeneity within the E6 ORF region of Iranian HPV16. Tsakogiannis et al. reported nine nucleotide variants A184G, G201A, G219A, A280T, T302A, A336G, G514A, C523T, C539T, G514A, and C539T in HPV16 E6 gene [33]. Pande et al. observed amino acid substitutions Q14H, H78Y, and L83V [7]. Boumba et al. in 2015 reported five typical mutations, C143G, G145T, T286A, A289G, and C335T at the E6 genomic region [34]. Assoumou et al. described all cases that exhibited at least one specific nucleotide variant in the E6 gene [35]. Results of our study are consistent with the earlier reports. Most interestingly, more than 90% distribution of the HPV16 E6 variant, T213G, was detected in our study; thus, it was found either alone 92.40% (73/79) or in combination with additional E6 gene mutations. We showed for the first time that the novel E6 gene mutation, G50T (R17I), observed in the 11.39% (9/79) Iranian HPV16-positive cases. These last mutations were located at the N-terminal domain of the E6 antigen.

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In another aspect of this study, the nucleotide sequence of the E6 ORF of HPV16 was optimized [36]. There exist some literature about codon optimization in L. lactis such as IL-2 (CAI: 0.22) and IL-6 (CAI 0.3) from Mus musculus, egg white lysozyme (CAI: 0.23), and Tetanus toxin fragment C (CAI: 0.33) from Gallus domesticus and Clostridium tetani, respectively, peptidase pep N (CAI: 0.55) and peptidase pep X (CAI: 0.49) from Lactobacillus helveticus and Urease ureB (CAI: 0.51) from Helicobacter pylori [37,38]. The data are significant in light of the CAI values of the naturally happening CAI values of genes in L. lactis. Hence, it becomes clear that CAI values of 0.3 or lower are very rare [39]. In the current study, the CAI value of HPV16 E6 gene improved from 0.264 to 1.000 using codon usage table of L. lactis subsp. cremoris subsp. MG1363. Another possibility is that codon optimization of the E6 gene resulted in reduced mRNA stability within L. lactis [40]. Attractively, it has also been extensively reported that codons with elevated G+C content, which permit the mRNA to fold into a stable secondary structure appear to inhibit translation initiation and decrease protein synthesis [41,42]. Since L. lactis is an AT-rich genome, it is estimated that A- and/or T-ending codons will dominate in the coding regions of this organism. HPV16 E6 gene has an average genomic G+C content of 37.70%. In opposition, the sequenced genomes of L. lactis subsp. cremoris MG1363 strain indicate that the specie has an average G+C content of 36.72% [43,44]. It is therefore unavoidable that disparity in the codon usage of HPV16 E6 gene and L. lactis would lead to inhibitory issues through heterologous protein expression [45]. Consequently, we thought that the codon-optimized HPV16 E6 synthetic gene not only helped from an augmented translational efficacy due to improved accessibility of cognate tRNAs for each codon over the native gene, but also that the attendant increase in A+U content at the 5 region reduced the mRNA stability, enhanced translation initiation and led to greater E6 synthesis. We clearly confirmed that the two forms of E6 and optiE6 were successfully induced in the recombinant NZ9000 strain of L. lactis using the pNZ8148 vector. Our results demonstrated that the nisin-induced L. lactis NZ9000 has the ability to recognize infrequent HPV16 E6 codons. The outcomes exhibited that the signal amounts of the band in L. lactis harboring pNZ8148-HPV16-E6 was noticeably weak and the band corresponded to the L. lactis having pNZ8148-HPV16-optiE6 was around approximately threefold higher than rE6 oncoprotein representing that codon optimization might synergistically progress expression of rE6 from an unnoticeable level to a noticeable level in the host L. lactis NZ9000. Our outcomes suggest that recombinant L. lactis harboring codon-optimized E6 oncogene of HPV16 may be a hopeful therapeutic oral vaccine candidate for the elimination of cervical cancer. Therefore, we intend in the future to investigate oral immunogenicity of recombinant bacteria in our patients. Conclusion To the best of our knowledge, this is the first study describing nucleotide variants and evolutionary pressure acting on E6 regions of HPV16 genomes from the Iranian population. In conclusion, the data described here is valuable for future investigation on oncogenic potential, and can offer critical data for developing diagnostic and as well as designing vaccines for a specific population. Acknowledgements The authors would like to gratefully acknowledge AH Mohseni for his assistance in bioinformatics analysis and his critic reading of the manuscript.

Financial & competing interests disclosure This work was supported by Keyvan Virology Specialty Laboratory (KVSL) in cooperation with the Islamic Azad University, Science and Research Branch (SRBIAU; Tehran, Iran). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.

Ethical conduct of research The authors state that they have obtained appropriate institutional review board approval or have followed the principles outlined in the Declaration of Helsinki for all human or animal experimental investigations. In addition, for investigations involving human subjects, informed consent has been obtained from the participants involved.


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Summary points r Codon optimization has been considered an effective methodology for increasing the expression levels of heterologous genes that have codons rarely used in the host microorganism. r These data suggest that codon optimization of oncogene could be important in making more efficacious DNA vaccine for tumor protection in the future investigation.

References

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1

Bosch FX, Manos MM, Muñoz N et al. Prevalence of human papillomavirus in cervical cancer: a worldwide perspective. J. Natl Cancer. Inst. 87(11), 796–802 (1995).

2

Bedell MA, Jones KH, Grossman SR, Laimins L. Identification of human papillomavirus type 18 transforming genes in immortalized and primary cells. J. Virol. 63(3), 1247–1255 (1989).

3

Pan H, Griep AE. Altered cell cycle regulation in the lens of HPV-16 E6 or E7 transgenic mice: implications for tumor suppressor gene function in development. Genes Dev. 8(11), 1285–1299 (1994).

4

Zehbe I, Wilander E, Delius H, Tommasino M. Human papillomavirus 16 E6 variants are more prevalent in invasive cervical carcinoma than the prototype. Cancer Res. 58(4), 829–833 (1998).

5

Van Duin M, Snijders PJ, Vossen MT et al. Analysis of human papillomavirus type 16 E6 variants in relation to p53 codon 72 polymorphism genotypes in cervical carcinogenesis. J. Gen. Virol. 81(2), 317–325 (2000).

6

Ho L, Chan S, Chow V et al. Sequence variants of human papillomavirus type 16 in clinical samples permit verification and extension of epidemiological studies and construction of a phylogenetic tree. J. Clin. Microbiol. 29(9), 1765–1772 (1991).

7

Pande S, Jain N, Prusty BK et al. Human papillomavirus type 16 variant analysis of E6, E7, and L1 genes and long control region in biopsy samples from cervical cancer patients in north India. J. Clin. Microbiol. 46(3), 1060–1066 (2008).

8

Poquet I, Ehrlich SD, Gruss A. An export-specific reporter designed for Gram-positive bacteria: application to Lactococcus lactis. J. Bacteriol. 180(7), 1904–1912 (1998).

9

Ravn P, Arnau J, Madsen SM, Vrang A, Israelsen H. The development of TnNuc and its use for the isolation of novel secretion signals in Lactococcus lactis. Gene 242(1), 347–356 (2000).

10

Maguin E, Prevost H, Ehrlich SD, Gruss A. Efficient insertional mutagenesis in lactococci and other Gram-positive bacteria. J. Bacteriol. 178(3), 931–935 (1996).

11

Bolotin A, Wincker P, Mauger S et al. The complete genome sequence of the lactic acid bacterium Lactococcus lactis ssp. lactis IL1403. Genome Res. 11(5), 731–753 (2001).

12

Dieye Y, Usai S, Clier F, Gruss A, Piard J-C. Design of a protein-targeting system for lactic acid bacteria. J. Bacteriol. 183(14), 4157–4166 (2001).

13

Bermúdez-Humarán LG, Langella P, Commissaire J et al. Controlled intra-or extracellular production of staphylococcal nuclease and ovine omega interferon in Lactococcus lactis. FEMS Microbiol. Lett. 224(2), 307–313 (2003).

14

Gustafsson C, Govindarajan S, Minshull J. Codon bias and heterologous protein expression. Trends Biotechnol. 22(7), 346–353 (2004).

15

Wu X, Jörnvall H, Berndt KD, Oppermann U. Codon optimization reveals critical factors for high level expression of two rare codon genes in Escherichia coli: RNA stability and secondary structure but not tRNA abundance. Biochem. Biophys. Res. Commun. 313(1), 89–96 (2004).

16

Outchkourov NS, Stiekema WJ, Jongsma MA. Optimization of the expression of equistatin in Pichia pastoris. Protein Expr. Purif. 24(1), 18–24 (2002).

17

De Rocher EJ, Vargo-Gogola TC, Diehn SH, Green PJ. Direct evidence for rapid degradation of Bacillus thuringiensis Toxin mRNA as a cause of poor expression in plants. Plant Physiol. 117(4), 1445–1461 (1998).

18

Kofman A, Graf M, Deml L, Wolf H, Wagner R. Codon usage-mediated inhibition of HIV-1 gag expression in mammalian cells occurs independently of translation. Tsitologiia 45(1), 94–100 (2002).

19

Koda A, Bogaki T, Minetoki T, Hirotsune M. High expression of a synthetic gene encoding potato α-glucan phosphorylase in Aspergillus niger. J. Biosci. Bioeng. 100(5), 531–537 (2005).

20

Keyvani H, Saroukalaei ST, Mohseni AH. Assessment of the human Cytomegalovirus UL97 gene for identification of resistance to ganciclovir in Iranian immunosuppressed patients. Jundishapur J. Microbiol. 9(5), e31733 (2016).

21

Kleter B, Van Doorn L-J, Ter Schegget J et al. Novel short-fragment PCR assay for highly sensitive broad-spectrum detection of anogenital human papillomaviruses. Am. J. Pathol. 153(6), 1731–1739 (1998).

22

Wright F. The ‘effective number of codons’ used in a gene. Gene 87(1), 23–29 (1990).

23

Comeron JM, Aguadé M. An evaluation of measures of synonymous codon usage bias. J. Mol. Evol. 47(3), 268–274 (1998).

24

Fuglsang A. The ‘effective number of codons’ revisited. Biochem. Biophys. Res. Commun. 317(3), 957–964 (2004).




25

Nakamura Y, Gojobori T, Ikemura T. Codon usage database. Nucleic Acids Res. 26, 334 (1998). http://www.kazusa.or.jp/codon

26

Puigbo P, Guzman E, Romeu A, Garcia-Vallve S. OPTIMIZER: a web server for optimizing the codon usage of DNA sequences. Nucleic Acids Res. 35(suppl 2), W126–W131 (2007).

27

Rasband W. ImageJ, US National Institutes of Health, MD, USA (1997). http.imagej.nih.gov/ij

28

Salehi-Vaziri M, Sadeghi F, Hashemi FS et al. Distribution of human papillomavirus genotypes in iranian women according to the severity of the cervical lesion. Iran Red Crescent Med. J. 18(4), e24458 (2016).

29

Haghshenas M, Golini-Moghaddam T, Rafiei A, Emadeian O, Shykhpour A, Ashrafi GH. Prevalence and type distribution of high-risk human papillomavirus in patients with cervical cancer: a population-based study. Infect. Agent. Cancer 8(1), 20 (2013).

30

Khodakarami N, Clifford GM, Yavari P et al. Human papillomavirus infection in women with and without cervical cancer in Tehran, Iran. Int. J. Cancer 131(2), E156–E161 (2012).

31

Ghaffari SR, Sabokbar T, Mollahajian H et al. Prevalence of human papillomavirus genotypes in women with normal and abnormal cervical cytology in Iran. Asian Pac. J. Cancer. Prev. 7(4), 529 (2006).

32

Bao YP, Li N, Smith JS, Qiao YL. Human papillomavirus type distribution in women from Asia: a meta-analysis. Int. J. Gynecol. Cancer 18(1), 71–79 (2008).

33

Tsakogiannis D, Papadopoulou A, Kontostathi G et al. Molecular and evolutionary analysis of HPV16 E6 and E7 genes in Greek women. J. Med. Microbiol. 62(11), 1688–1696 (2013).

34

Boumba LMA, Assoumou SZ, Hilali L, Mambou JV, Moukassa D, Ennaji MM. Genetic variability in E6 and E7 oncogenes of human papillomavirus Type 16 from Congolese cervical cancer isolates. Infect. Agent Cancer. 10(1), 15 (2015).

35

Assoumou SZ, Boumba LMA, Mbiguino AN et al. Sequence variations of human Papillomavirus Type 16 E6 and E7 genes in cervical cancer isolates from Gabon. Br. Microbiol. Res. J. 8(2), 386–394 (2015).

36

Stenström CM, Holmgren E, Isaksson LA. Cooperative effects by the initiation codon and its flanking regions on translation initiation. Gene 273(2), 259–265 (2001).

37

Luoma S, Peltoniemi K, Joutsjoki V et al. Expression of six peptidases from Lactobacillus helveticus in Lactococcus lactis. Appl. Environ. Microbiol. 67(3), 1232–1238 (2001).

38

Tuler T, Callanan M, Klaenhammer T. Overexpression of peptidases in Lactococcus and evaluation of their release from leaky cells. J. Dairy. Sci. 85(10), 2438–2450 (2002).

39

Fuglsang A. Lactic acid bacteria as prime candidates for codon optimization. Biochem. Biophys. Res. Commun. 312(2), 285–291 (2003).

40

Deana A, Belasco JG. Lost in translation: the influence of ribosomes on bacterial mRNA decay. Genes Dev. 19(21), 2526–2533 (2005).

41

Angov E. Codon usage: nature’s roadmap to expression and folding of proteins. Biotechnol. J. 6(6), 650–659 (2011).

42

Gu W, Zhou T, Wilke CO. A universal trend of reduced mRNA stability near the translation-initiation site in prokaryotes and eukaryotes. PLoS Comput Biol 6(2), e1000664 (2010).

43

Claesson MJ, Li Y, Leahy S et al. Multireplicon genome architecture of Lactobacillus salivarius. Proc. Natl Acad. Sci. 103(17), 6718–6723 (2006).

44

Cho Y-J, Choi JK, Kim J-H et al. Genome sequence of Lactobacillus salivarius GJ-24, a probiotic strain isolated from healthy adult intestine. J. Bacteriol. 193(18), 5021–5022 (2011).

45

Johnston C, Douarre PE, Soulimane T et al. Codon optimisation to improve expression of a Mycobacterium avium ssp. paratuberculosis-specific membrane-associated antigen by Lactobacillus salivarius. Pathog. Dis. 68(1), 27–38 (2013).


Research Article

10.2217/fvl-2017-0032