Codon Optimized Hpv16 Li For Salmonella Vaccine Strains Against Human Papillomavirus Type 16
Abstract:
The present invention relates to a novel nucleic acid sequence (HPV16 L1S) encoding antigenic HPV16 LI protein as provided in SEQ ID NO: 1, wherein the said sequence has at least one modified codon for optimum stability of recombinant plasmid vector when transformed in the prokaryotic micro-organism for improved immunogenicity of the resulting prokaryotic micro-organism. The invention further relates to constructing recombinant vectors pFS14nsdHPV16Ll and pFS14nsdHPV16 kan L1S harboring SEQ ID NO: 1, wherein the former carries Ampicillin and the latter, Kanamycin as a selection marker. The invention also relates to an attenuated strain of a prokaryotic micro-organism transformed with nucleic acid encoding HPV16 (Human Papillomavirus) major capsid protein and expressing the corresponding protein. In addition the invention discloses a process of producing a vaccine based on prokaryotic micro-organism for the treatment of papillomavirus infection and associated risk of cancer.
Get Free WhatsApp Updates!
Notices, Deadlines & Correspondence
DEPT. OF GYNECOLOGY-OBSTERICS, HOSPICES/CHUV 21,RUE,DU BUGNON CH-1005 LAUSANNE,
Specification
CODON-OPTIMIZED HPV16 LI FOR SALMONELLA VACCINE STRAINS AGAINST HUMAN PAPILLOMAVIRUS TYPE 16
TECHNICAL FIELD
The present invention relates to a novel nucleic acid sequence (HPV 16 L1S) encoding antigenic HPV 16 LI protein as provided in SEQ ID NO: I, wherein the said sequence has atleast one modified codon for optimum stability of recombinant plasmid vector when transformed in the prokaryotic micro-organism for improved immunogenicity of the resulting prokaryotic micro-organism. The invention also relates to an attenuated strain of a prokaryotic micro-organism transformed with nucleic acid encoding HPV 16 (Human Papillomavirus) major capsid protein and expressing the corresponding protein. In addition the invention discloses a process of producing a vaccine based on prokaryotic micro-organism for the treatment of papillomavirus infection and associated risk of cancer.
BACKGROUND AND PRIOR ART REFERENCES
Cervical cancer is the second leading cause of cancer deaths in women worldwide, and virtually all of these tumors are attributable to infection with a sub-set of human papillomaviruses (HPVs), of which HPV16 is found most frequently (6, 41). An effective vaccine against these HPVs would therefore be expected to have a dramatic impact on the incidence of this cancer and its precursor lesions, as well as on the less common tumors attributable to these viruses. The leading candidate is a prophylactic sub-unit HPV virus-like particle (VLP) vaccine (reviewed by (35) and (24)). A proof of principle efficacy trial showed that women vaccinated with HPV 16 VLPs were highly protected against genital mucosal infection by this viral type (19). However, the requirement for multiple injections for a vaccine whose anticipated target population will be older than those of childhood vaccines may represent a substantial hurdle for widespread implementation. This is particularly true in the developing world, which accounts for more than three-quarters of the worldwide cases of cervical cancer (6), Recombinant attenuated Salmonella strains that are attenuated yet invasive have been widely used as mucosal vaccine vectors to deliver pathogen-specific protective epitopes into the mucosal-associated lymphoid tissues. Via this route, both mucosal and systemic immune responses against the carrier and the foreign antigens may be obtained (reviewed in (11, 22, 36)). We have shown that nasal vaccination of mice with Salmonella expressing the HPV 16 major capsid protein LI, that self-assembles
into VLPs, induces anti-HPV16 conformational and neutralizing antibodies in serum and genital secretions provided the attenuated Salmonella enterica serovar Typhimurium strains exhibit the PhoPc phenotype (3, 4, 31). However, even with the original PhoPc strain, a double nasal immunization was required to induce high anti-HPV16 VLP antibody liters while oral immunization was inefficient (31). The observations of low levels of LI expression together with a high instability of the Ll-encoding plasmids in absence of antibiotic selection strongly suggested that either the LI protein or the Ll gene could be toxic to the bacteria. As the viral Ll gene exhibits a highly unfavorable codon usage for expression in Salmonella, we designed and tested herein a synthetic nucleotide sequence (referred as L1S hereafter) encoding the Ll protein with optimized codons for translation in Salmonella.
The HPV vaccines based on such VLPs and currently tested in clinical trials have proven to be well tolerated, highly immunogenic, and able to prevent the development of HPV16-induced cervical intraepithelial neoplasia (reviewed by [Schiller, 2004 #1431] and [Lowy, 2003 #1397]). However, these expensive vaccines require multiple intramuscular doses to be efficient and since most of cervical cancers occur in developing countries, such vaccines appear to be inaccessible for people needing them most. It is thus of great importance to develop other strategies that have world wide applicability. Live attenuated Salmonella strains may be effective antigen delivery systems, as they are able to express foreign antigens and to elicit mucosal as well as systemic immune responses against homologous and heterologous antigens after oral vaccination (reviewed in [Schddel, 1992 #245; Curtiss, 1994 #466; Levine, 1997 #1416]. In this study, we have further optimized the Salmonella-based vaccine against HPV 16 so that it could be tested in women. We have first replaced the ampicilin selection marker used for plasmid maintenance by a kanamycine resistance gene that is more acceptable for use in human given its higher biosafety record (FDA approved in 1994 [Administration, 1994 #1522]). Then, we have tested the new plasmid in three Salmonella enterica serovar Typhi vaccine strains that have been shown to be safe in human, i.e. Ty21a [Germanier, 1975 #103], the actual licensed typhoid vaccine, as well as Ty800 [Hohmann, 1996 #596] and CVD908htrA [Tacket, 1997 #643, two highly immunogenic typhoid candidate vaccines. Using a model of intranasal immunisation of mice [Galen, 1997 #661], the immune responses elicited by these three strains against the homologous and heterologous antigens were compared. Our data show that anti-HVP16 YLP humoral responses after either nasal or oral immunization
with the new recombinant strains were highly increased. Interestingly, this was not associated with an increased LI expression, but with a remarkable stability of the L1S-expressing plasmid in vitro and in vivo. In addition, immunogenicity was not restricted to PhoPc, as shown with other Salmonella enterica serovar Typhimurium strains whose attenuations are suitable for human use.
BRIEF DESCRIPTION OF ACCOMPANYING DRAWINGS
Figure 1 shows Codon-optimized HPV16 LIS or of. (SEQ ID NO: l)The nucleotide sequence of LIS is shown with the modified codons underlined and modified nucleotides in bold.
Figure 2 shows HPV16L1 expression in PhoP0 LI and PhoPc LIS recombinant strains. Figure 3 shows LI and LIS-plasmid stability in vitro. Figure 4 shows Anti-HPV16 VLP systemic (A) and vaginal (B) antibody titers after nasal and
oral vaccination with PhoPc LIS. Figure 5 shows Anti-HPV16 VLP systemic IgG titers after nasal or oral vaccination with
%4989 LIS, x4990 LIS, PhoP" LIS and AroA LIS. Figure 6 is a comparison of serum anti-HPV16 VLP antibody titers after oral vaccination
with PhoPc kan LIS, Phop" kan LIS and AAro A kan LIS. Figure 7 shows in vitro stability of the kan LIS plasmid in different Salmonella enterica
serovar Typhi strains. Figure 8 is comparison of serum anti-HPV16 VLP antibody titers after nasal vaccination
with Ty21a kan LIS, Ty800 kan LIS and CVD908htrA kan LIS.
Figure 9 is comparison of HPV16 VLP and Flagellin -specific CD4+ T cell proliferations. Figure 10 shows HPV16-neutraIizing and anti-HPV16 VLP antibodies in serum and
vaginal secretions of mice nasally vaccinated with Ty21a kan LIS.
Figure 11 shows HPV16 neutralizing and anti-HPV16 VLP antibodies in serum and vaginal secretions of mice nasally vaccinated with Ty21a kan LIS alone or primed with purified VLPs.
DETAILS OF TABLES REFERRED IN THE SPECIFICATION
TABLE 1 & 1A refers to Salmonella strains used in this study
TABLE 2 refers to recovery of Salmonella PhoPc earring LI- or LlS-encoding plasmids
two weeks after nasal or oral immunization TABLE 2A refers to recovery of Salmonella PhoPc LlS-encoding plasmids carrying the
ampiciline or the kanamycine resistant genes two weeks after oral immunization
TABLE 3 refers to recovery of different Salmonella enterica serovar Typhi carrying kan L1S plasmids one week after nasal immunization.
OBJECTS OF THE INVENTION
The main object of the present invention is to provide a novel nucleic acid sequence (HPV16 L1S) encoding antigenic HPV16 LI protein as provided in SEQ ID NO: 1, wherein said sequence has at least one modified codon for optimum stability of recombinant plasmid vector when transformed in the prokaryotic micro-organism for improved immunogenicity of the resulting prokaryotic micro-organism.
Another object of the present invention is to construct recombinant vectors pFS14nsdHPV16Ll, pFS14nsdHPV16 kan L1S and pFSnsd-kan HinS-HPV16LlS harboring SEQ ID NO: 1, wherein the former carries Ampicillin and the latter, Kanamycin as a selection marker.
Yet another object of the invention provides an attenuated strain of a prokaryotic micro-organism transformed with nucleic acid encoding HPV16 (Human Papillomavirus) major capsid protein and expressing the corresponding protein.
One more object of the invention is to provide a process of producing a vaccine based on prokaryotic micro-organism for the treatment of papillomavirus infection and associated risk of cancer.
SUMMARY OF INVENTION
Accordingly, the present invention provides a novel nucleic acid sequence (HPV16 L1S) as provided in SEQ ID NO: 1, based on Human Papilloma Virus Type 16 (HPV 16) encoding antigenic HPV 16 LI protein, wherein the said sequence has at least one modified codon for optimum stability of recombinant plasmid vector when transformed in the prokaryotic micro-organism for improved immunogenicity of the resulting prokaryotic micro-organism, preferably the strains belonging to Salmonella species. The invention further relates to constructing recombinant vectors pFS14nsdHPV16Ll, pFS14nsdHPV16 kan L1S and pFSnsd-kan HinS-HPV16LlS harboring SEQ ID NO: 1, wherein the former carries Ampicillin and the latter, Kanamycin as a selection marker. The invention further provides an attenuated strain belonging to Salmonella species transformed with pFS14nsdHPV16Ll, pFS14nsdHPV16 kan L1S or pFSnsd-kan HinS-HPV16LlS harboring SEQ ID NO: 1. In addition the invention discloses a process for producing a
Salmonella based vaccine for the treatment of papillomavirus infection and associated risk of cancer.
DETAILED DESCRIPTION OF THE INVENTION
The development of a Salmonella-based vaccine against H-PV infection and associated lesions would be of great value for a worldwide implementation with the theoretical advantage to induce long-lasting systemic and mucosal immunity with a single oral vaccination. However, despite we showed the feasibility of such a strategy in mice (31), several drawbacks had to be addressed before a Salmonella-based vaccine could be safely tested in women. Those drawbacks included the requirement of a particular Salmonella phenotype (PhoPc, (3, 4)) and the nasal route of immunization to efficiently induce neutralizing antibody responses, as well as the observation that the LI-encoding plasmid was unstable without antibiotic selection (31, 4) or poorly expressed when stabilized with a semi-lethal complementation system (3). Here we report that most of these problems are solved by using a codon-optimization strategy for the expression of the HPV16 LI capsid gene (HPV16L1S). Indeed, expression of the synthetic LIS gene is stable in Salmonella and results in higher immunogenicity when differently attenuated bacteria are delivered by either the nasal or oral route.
Expression of native papillomavirus capsid genes is limited in mammalian cells, but the resulting lack of immunogenicity of HPV DNA vaccines could be relieved by codon optimization (20, 23 and 42). The influence of codon usage on the immunogenicity has been recognized for other DNA vaccines (1, 12, 32 and 38), where higher expression of the heterologous genes resulted in higher immunogenicity. As the codon usage of the original HPV 16 capsid gene is also suboptimal for translation in Salmonella, we anticipated that expression of a codon-optimized LIS gene would result in higher VLP expression and consequently higher immunogenicity of the recombinant Salmonella. To our surprise, the higher immunogenicity of the differently attenuated LIS recombinant Salmonella does not correlate with higher amounts of Ll/VLPs produced in these bacteria. In fact, the opposite is true and lower amounts of HPV 16 VLPs were produced when the LIS gene was expressed
(ranging from ca. 3μg /1011 CFU for the AroA LIS strain to 23|μg /1011 CFU for x4989 LIS) as compared to the expression of the original LI sequence (VLP amounts between 20 and 60μg/10n CFU, (4)). This is in contrast to the >104 increase in LI expression obtained in mammalian cells with a human-optimized HPV16 LI gene (20). We should note
however, that we cannot exclude that the amounts of VLPs expressed in the bacteria may vary when the Salmonella are invading the mouse tissues where the metabolic constraints are different. Unfortunately, we are unable to measure VLP expression in vivo, given the relatively low number of bacteria recovered (103-104 CFU/organ) and the low VLP expression achieved (
Documents
Application Documents
#
Name
Date
1
131-chenp-2007-sequence listing.pdf
2011-09-02
1
131-CHENP-2007_EXAMREPORT.pdf
2016-07-02
2
131-CHENP-2007 AMENDED CLAIMS 19-10-2011.pdf
2011-10-19
2
131-chenp-2007-pct.pdf
2011-09-02
3
131-chenp-2007-form 5.pdf
2011-09-02
3
131-CHENP-2007 AMENDED PAGES OF SPECIFICATION 19-10-2011.pdf
2011-10-19
4
131-chenp-2007-form 3.pdf
2011-09-02
4
131-CHENP-2007 FORM-3 19-10-2011.pdf
2011-10-19
5
131-chenp-2007-form 1.pdf
2011-09-02
5
131-CHENP-2007 FORM-5 19-10-2011.pdf
2011-10-19
6
131-chenp-2007-drawings.pdf
2011-09-02
6
131-CHENP-2007 POWER OF ATTORNEY 19-10-2011.pdf
2011-10-19
7
131-chenp-2007-description(complete).pdf
2011-09-02
7
131-CHENP-2007 EXAMINATION REPORT REPLY RECEIVED 19-10-2011.pdf
2011-10-19
8
131-chenp-2007 correspondence others.pdf
2011-09-02
8
131-chenp-2007-correspondnece-others.pdf
2011-09-02
9
131-chenp-2007-claims.pdf
2011-09-02
10
131-chenp-2007-abstract.pdf
2011-09-02
11
131-chenp-2007-claims.pdf
2011-09-02
12
131-chenp-2007 correspondence others.pdf
2011-09-02
12
131-chenp-2007-correspondnece-others.pdf
2011-09-02
13
131-CHENP-2007 EXAMINATION REPORT REPLY RECEIVED 19-10-2011.pdf
2011-10-19
13
131-chenp-2007-description(complete).pdf
2011-09-02
14
131-CHENP-2007 POWER OF ATTORNEY 19-10-2011.pdf
2011-10-19
14
131-chenp-2007-drawings.pdf
2011-09-02
15
131-CHENP-2007 FORM-5 19-10-2011.pdf
2011-10-19
15
131-chenp-2007-form 1.pdf
2011-09-02
16
131-CHENP-2007 FORM-3 19-10-2011.pdf
2011-10-19
16
131-chenp-2007-form 3.pdf
2011-09-02
17
131-CHENP-2007 AMENDED PAGES OF SPECIFICATION 19-10-2011.pdf