Patent Application
20070172930
This invention relates to a process for the selection of HIV-1 subtype (clade) C isolates, selected HIV-1 subtype C isolates, their genes and modifications and derivatives thereof for use in prophylactic and therapeutic vaccines to produce proteins and polypeptides for the purpose of eliciting protection against HIV infection or disease.
The disease acquired immunodeficiency syndrome (AIDS) is caused by human immunodeficiency virus (HIV). Over 34 million people worldwide are thought to be living with HIV/AIDS, with over 90% of infected people living in developing countries (UNAIDS, 1999). It is estimated that 24 million infected people reside in sub-Saharan Africa and that South Africa currently has one of the world's fastest growing HIV-1 epidemics. At the end of 1999, over 22% of pregnant women attending government antenatal clinics in South Africa were HIV positive (Department of Health, 2000). A preventative vaccine is considered to be the only feasible way to control this epidemic in the long term.
HIV shows remarkable genetic diversity that has confounded the development of a vaccine. The molecular basis of variation resides in the viral enzyme reverse transcriptase which not only introduces an error every round of replication, but also promotes recombination between viral RNAs. Based on phylogenetic analysis of sequences, HIV has been classified into a number of groups: the M (major group) which comprises subtypes A to H and K, the O (outlier group) and the N (non-M, non-O group). Recently recombinant viruses have been more frequently identified and there are a number which have spread significantly and established epidemics (circulating recombinant forms or CRF) such as subtype A/G recombinant in West Africa, and CRF A/E recombinant in Thailand (Robertson et al., 2000).
Subtype C predominates in the Southern African region which includes Botswana, Zimbabwe, Zambia, Malawi, Mozambique and South Africa. In addition, increasing numbers of subtype C infections are being detected in the Southern region of Tanzania. This subtype also predominates in Ethiopia and India and is becoming more important in China.
A possible further obstacle to vaccine development is that the biological properties of HIV change as disease progresses. HIV requires two receptors to infect cells, the CD4 and co-receptors of which CCR5 and CXCR4 are the major co-receptors used by HIV-1 strains. The most commonly transmitted phenotype is non-syncytium inducing (NSI), macrophage-tropic viruses that utilise the CCR5 co-receptor for entry (R5 viruses). Langerhans cells in the mucosa are thought to selectively pick up R5 variants at the portal of entry and transport them to the lymph nodes where they undergo replication and expansion. As the infection progresses, viruses evolve that have increased replicative capacity and the ability to grow in T cell lines. These syncytium-inducing (SI) T-tropic viruses use CXCR4 in conjunction with or in preference to CCR5, and in some cases also use other minor co-receptors (Connor et al., 1997, Richman & Bozzette, 1994). However HIV-1 subtype C viruses appear to be unusual in that they do not readily undergo this phenotypic switch, as R5 viruses are also predominant in patients with advanced AIDS (Bjorndal et al., 1999, Peeters et al., 1999, Ping et al., 1999, Tscherning et al., 1998, Scarlatti et al., 1997).
According to one aspect of the invention a process for the selection of HIV subtype isolates for use in the development of prophylactic and therapeutic pharmaceutical composition comprises the following steps: isolating viruses from recently infected subjects; generating a consensus sequence for at least part of at least one HIV gene by identifying the most common codon or amino acid among the isolated viruses at each position along at least part of the gene; and selecting the isolated virus or viruses with a high sequence identity to the consensus sequence, a phenotype which is associated with transmission for the particular HIV subtype.
The isolated virus may be of the same subtype as a likely challenge strain.
The HIV subtype is preferably HIV-1 subtype C.
For HIV-1 subtype C, the phenotype which is associated with transmission is typically a virus that utilizes the CCR5 co-receptor and is non syncitium inducing (NSI).
According to another aspect of the invention an HIV-1 subtype C isolate, designated Du422 and assigned Provisional Accession Number 01032114 by the European Collection of Cell Cultures, is provided.
According to another aspect of the invention an HIV-1 subtype C isolate, designated Du151 and assigned Accession Number 00072724 by the European Collection of Cell Cultures, is provided.
According to another aspect of the invention an HIV-1 subtype C isolate, designated Du179 and assigned Accession Number 00072725 by the European Collection of Cell Cultures, is provided.
According to another aspect of the invention a molecule is provided, the molecule having: (i) the nucleotide sequence set out in sequence as set out in SEQ ID NO: 1; (ii) an RNA sequence corresponding to the nucleotide sequence set out in SEQ ID NO: 1; (iii) a sequence which will hybridize to the nucleotide sequence set out in SEQ ID NO: 1 or an RNA sequence corresponding to it, under strict hybridization conditions; (iv) a sequence which is homologous to the nucleotide sequence set out in SEQ ID NO: 1 or an RNA sequence corresponding to it; or (v) a sequence which is a modification or derivative of the sequence of any one of (i) to (iv).
The modified sequence is preferably that set out in SEQ ID NO: 7.
According to another aspect of the invention a molecule is provided, the molecule having: (i) the nucleotide sequence set out in SEQ ID NO: 3; (ii) an RNA sequence corresponding to the nucleotide sequence set out in SEQ ID NO: 3; (iii) a sequence which will hybridize to the nucleotide sequence set out in SEQ ID NO: 3 or an RNA sequence corresponding to it, under strict hybridization conditions; (iv) a sequence which is homologous to the nucleotide sequence set out in SEQ ID NO: 3 or an RNA sequence corresponding to it; or (v) a sequence which is a modification or derivative of the sequence of any one of (i) to (iv).
The modified sequence is preferably that set out in SEQ ID NO: 9.
According to another aspect of the invention a molecule is provided, the molecule having: (i) the nucleotide sequence set out in SEQ ID NO: 5; (ii) an RNA sequence corresponding to the nucleotide sequence set out in SEQ ID NO: 5; (iii) a sequence which will hybridize to the nucleotide sequence set out in SEQ ID NO: 5 or an RNA sequence corresponding to it, under strict hybridization conditions; (iv) a sequence which is homologous to the nucleotide sequence set out in SEQ ID NO: 5 or an RNA sequence corresponding to it; or (v) a sequence which is a modification or derivative of the sequence of any one of (i) to (iv). The modified sequence is preferably that set out in SEQ ID NO: 11.
According to another aspect of the invention a molecule is provided, the molecule having: (i) the nucleotide sequence set out in SEQ ID NO: 13; (ii) an RNA sequence corresponding to the nucleotide sequence set out in SEQ ID NO: 13; (iii) a sequence which will hybridize to the nucleotide sequence set out in SEQ ID NO: 13 or an RNA sequence corresponding to it, under strict hybridization conditions; (iv) a sequence which is homologous to the nucleotide sequence set out in SEQ ID NO: 13 or an RNA sequence corresponding to it; or
(v) a sequence which is a modification or derivative of the sequence of any one of (i) to (iv).
The modified sequence preferably has similar or the same modifications as those set out in SEQ ID NO: 11 for the env gene of the isolate Du151.
According to another aspect of the invention a polypeptide is provided, the polypeptide having: (i) the amino acid sequence set out in SEQ ID NO: 2; or (ii) a sequence which is a modification or derivative of the amino acid sequence set out in SEQ ID NO: 2.
The modified sequence is preferably that set out in SEQ ID NO: 8.
According to another aspect of the invention a polypeptide is provided, the polypeptide having: (i) the amino acid sequence set out in SEQ ID NO: 4; or (ii) a sequence which is a modification or derivative of the amino acid sequence set out in SEQ ID NO: 4. The modified sequence is preferably that set out in SEQ ID NO: 10.
According to another aspect of the invention a polypeptide is provided, the polypeptide having: (i) the amino acid sequence set out in SEQ ID NO: 6; or (ii) a sequence which is a modification or derivative of the amino acid sequence set out in SEQ ID NO: 6.
The modified sequence is preferably that set out in SEQ ID NO: 12.
According to another aspect of the invention a polypeptide is provided, the polypeptide having: (i) the amino acid sequence set out in SEQ ID NO: 14; (ii) a sequence which is a modification or derivative of the amino acid sequence set out in SEQ ID NO: 14.
The modified sequence preferably has similar or the same modifications as those set out in SEQ ID NO: 12 for the amino acid sequence of the env gene of the isolate Du151.
According to another aspect of the invention a consensus amino acid sequence for the partial gag gene of HIV-1 subtype C is the following:
According to another aspect of the invention a consensus amino acid sequence for the partial pol gene of HIV-1 subtype C is the following:
According to another aspect of the invention a consensus amino acid sequence for the partial env gene of HIV-1 subtype C is the following:
SEQ ID NO: 1 shows the nucleic acid sequence (cDNA) of the sequenced gag gene of the isolate Du422;
SEQ ID NO: 2 shows the amino acid sequence of the sequenced gag gene of the isolate Du422, derived from the nucleic acid sequence;
SEQ ID NO: 3 shows the nucleic acid sequence (cDNA) of the sequenced pol gene of the isolate Du151;
SEQ ID NO: 4 shows the amino acid sequence of the sequenced pol gene of the isolate Du151, derived from the nucleic acid sequence;
SEQ ID NO: 5 shows the nucleic acid sequence (cDNA) of the sequenced env gene of the isolate Du151;
SEQ ID NO: 6 shows the amino acid sequence of the sequenced env gene of the isolate Du151, derived from the nucleic acid sequence;
SEQ ID NO: 7 shows the nucleic acid sequence (DNA) of the resynthesized sequenced gag gene of the isolate Du422 modified to reflect human codon usage for the purposes of increased expression;
SEQ ID NO: 8 shows the amino acid sequence of the resynthesized sequenced gag gene of the isolate Du422 modified to reflect human codon usage for the purposes of increased expression;
SEQ ID NO: 9 shows the nucleic acid sequence (DNA) of the resynthesized sequenced pol gene of the isolate Du151 modified to reflect human codon usage for the purposes of increased expression;
SEQ ID NO: 10 shows the amino acid sequence of the resynthesized sequenced pol gene of the isolate Du151 modified to reflect human codon usage for the purposes of increased expression;
SEQ ID NO: 11 shows the nucleic acid sequence (DNA) of the resynthesized sequenced env gene of the isolate Du151 modified to reflect human codon usage for the purposes of increased expression;
SEQ ID NO: 12 shows the amino acid sequence of the resynthesized sequenced env gene of the isolate Du151 modified to reflect human codon usage for the purposes of increased expression;
SEQ ID NO: 13 shows the nucleic acid sequence (cDNA) of the sequenced env gene of the isolate Du179; and SEQ ID NO: 14 shows the amino acid sequence of the sequenced env gene of the isolate Du179.
This invention relates to the selection of HIV-1 subtype isolates and the use of their genes and modifications and derivatives thereof in making prophylactic and therapeutic pharmaceutical compositions and formulations, and in particular vaccines against HIV-1 subtype C. The compositions could therefore be used either prophylactically to prevent infection or therapeutically to prevent or modify disease. A number of factors must be taken into consideration in the development of an HIV vaccine and one aspect of the present invention relates to a process for the selection of suitable HIV isolates for the development of a vaccine.
The applicant envisages that the vaccine developed according to the above method could be used against one or more HIV subtypes other than HIV-1 subtype C.
An HIV vaccine aims to elicit both a CD8+ cytotoxic T lymphocyte (CTL) immune response as well as a neutralizing antibody response. Many current vaccine approaches have primarily focused on inducing a CTL response. It is thought that the CTL response may be more important as it is associated with the initial control of viral replication after infection, as well as control of replication during disease, and is inversely correlated with disease progression (Koup et al., 1994, Ogg et al., 1999 Schmitz et al., 1999). The importance of CTL in protecting individuals from infection is demonstrated by their presence in highly exposed seronegative individuals such as sex-workers (Rowland-Jones et al., 1998).
Knowledge of genetic diversity is highly relevant to the design of vaccines aiming at eliciting a cytotoxic T-lymphocyte (CTL) response. There are many CTL epitopes in common between viruses, particularly in the gag and pol region of the genome (HIV Molecular Immunology Database, 1998). In addition, several studies have now shown that there is a cross-reactive CTL response: individuals vaccinated with a subtype B-based vaccine could lyse autologous targets infected with a diverse group of isolates (Ferrari et al., 1997); and CTLs from non-B infected individuals could lyse subtype B-primed targets (Betts et al. 1997; Durali et al, 1998). A comparison of CTL epitopes in the HIV-1 sequence database shows about 40% of gp41 and 84% of p24 epitopes are identical or have only one amino acid difference between subtypes. Although this is a very crude analysis and does not take into consideration populations or dominant responses to certain epitopes, it does however indicate that there is a greater conservation of cytotoxic T epitopes within a subtype compared to between subtypes and that there will be a greater chance of a CTL response if the challenge virus is the same subtype as the vaccine strain.
The importance of genetic diversity in inducing a neutralizing antibody response appears to be less crucial. In general, neutralization serotypes are not related to genetic subtype. Some individuals elicit antibodies that can neutralize a broad range of viruses, including viruses of different subtypes while others fail to elicit effective neutralizing antibodies at all (Wyatt and Sodroski, 1998; Kostrikis et al., 1996; Moore et al., 1996). As neutralizing antibodies are largely evoked against functional domains of the virus which are essentially conserved, it is probable that HIV-1 genetic diversity may not be relevant in producing a vaccine designed to elicit neutralizing antibodies.
Viral strains used in the design of a vaccine need to be shown by genotypic analysis to be representative of the circulating strains and not an unusual or outlier strain. In addition, it is important that a vaccine strain also has the phenotype of a recently transmitted virus, which is NSI and uses the CCR5 co-receptor.
A process was developed to identify appropriate strains for use in developing a vaccine for HIV-1 subtype C. Viral isolates from acutely infected individuals were collected. They were sequenced in the env, gag and pol regions and the amino acid sequences for the env, gag and pol genes from these isolates were compared. A consensus sequence, the South African consensus sequence, was then formed by selecting the most frequently appearing amino acid at each position. The consensus sequence for each of the gag, pol and env genes of HIV-1 subtype C also forms an aspect of the invention. Appropriate strains for vaccine development were then selected from these isolates by comparing them with the consensus sequence and characterizing them phenotypically. The isolates also form an aspect of the invention.
In order to select for NSI strains which use the CCR5 co-receptor, a well established sex worker cohort was used to identify the appropriate strains. Appropriate strains were identified from acutely infected individuals by comparing them with the consensus sequence which had been formed. Viral isolates from fifteen acutely infected individuals were sequenced in the env, gag and pol and phenotypically characterized. These sequences were compared with viral isolates from fifteen asymptomatic individuals from another region having more than 500 CD4 cells and other published subtype C sequences located in the Los Alamos Database (http)://ww.hiv-web.lanl.gov/).
Three potential vaccine strains, designated Du151, Du422 and Du179, were selected. Du 151 and Du 422 were selected based on amino acid homology to the consensus sequence in all three gene regions env, gag and pol, CCR5 tropism and ability to grow and replicate in tissue culture. Du 179 is a R5X4 virus and was selected because the patient in which this strain was found showed a high level of neutralizing antibodies. The nucleotide and amino acid sequences of the three gene regions of the three isolates and modifications and derivatives thereof also form aspects of the invention.
The vaccines of the invention will be formulated in a number of different ways using a variety of different vectors. They involve encapsulating RNA or transcribed DNA sequences from the viruses in a variety of different vectors. The vaccines will contain at least part of the gag gene from the Du422 isolate, and at least part of the pol and env genes from the Du151 isolate of the present invention and/or at least part of the env gene from the Du179 isolate of the present invention or derivatives or modifications thereof.
Genes for use in DNA vaccines have been resynthesized to reflect human codon usage. The gag Du422 gene was designed so that the myristylation site and inhibitory sequences were removed. Similarly resynthesized gp 160 (the complete env gene consisting of gp 120 and gp 41) and pol genes will be expressed by DNA vaccines. The gp160 gene sequence has also been changed as described above for the gag gene to reflect human codon usage and the rev responsive element removed. The protease, inactivated reverse transcriptase and start of the RNAse H genes from Du151 pol are optimized for increased expression and will be joined with gag at an inserted Bgl1 site. The gag-pol frameshift will be maintained to keep the natural balance of gag to pol protein expression.
Another vaccine will contain DNA transcribed from the RNA for the gag gene from the Du422 isolate and RNA from the pol and env genes from the Du151 isolate and/or RNA from the env gene from the Du179 isolate. These genes could also be expressed as oligomeric envelope glycoprotein complexes (Progenics, USA) as published in J Virol 2000 January;74(2):627-43 (Binley, J. L. et al.), the adeno associated virus (AAV) (Target Genetics) and the Venezuelan equine encephalitus virus (U.S. patent application U.S. S No. 60/216,995, which is incorporated herein by reference).
The Isolation and Selection of Viral Strains for the Design of a Vaccine
The following criteria were used to select appropriate strains for inclusion into HIV-1 vaccines for Southern Africa:
that the strains be genotypically representative of circulating strains;
that the strain not be an outlier strain;
that the strain be as close as possible to the consensus amino acid sequence developed according to the invention for the env, gag and pol genes of HIV-1 subtype C;
that the strain have an R5 phenotype, i.e. a phenotype associated with transmission for selection of the RNA or cDNA to be included for the env region; and
that the vaccine be able to be grown in tissue culture.
The following procedure was followed in the selection of viral strains for the design of a vaccine. A well-established sex worker cohort in Kwazulu Natal, South Africa was used to identify the appropriate strains for use in an HIV vaccine. Viral isolates from 15 acutely infected individuals were sequenced in env, gag and pol and were also isolated and phenotypically characterized. These sequences were compared with a similar collection from asymptomatic individuals from the Gauteng region in South Africa as well as other published subtype C sequences.
Patients
Individuals with HIV infection were recruited from 4 regions in South Africa. Blood samples were obtained from recently infected sex workers from Kwazulu-Natal (n=13). Recent infection was defined as individuals who were previously seronegative and had became seropositive within the previous year. Samples were also collected from individuals attending out-patients clinics in Cape Town (n=2), women attending ante-natal clinics in Johannesburg (n=7) and men attending a STD clinic on a gold mine outside Johannesburg (n=8). The latter 2 groups were clinically stable and were classified as asymptomatic infections. Blood samples were collected in EDTA and used to determine the CD4 T cell count and genetic analysis of the virus. In the case of recent infections a branched chain (bDNA) assay (Chiron) to measure plasma viral load was done, and the virus was isolated. HIV-1 serostatus was determined by ELISA. The results of the CD4 T cell counts and the viral loads on the sex workers were established and information on the clinical status as at date of seroconversion, CD4, and data on the co-receptor usage is set out in Table 1 below.
Virus Isolation
HIV was isolated from peripheral blood mononuclear cells (PBMC) using standard co-culture techniques with mitogen-activated donor PBMC. 2.times.10.sup.6 patient PBMC were co-cultured with 2×106 donor PBMC in 12 well plates with 2 ml RPMI 1640 with 20% FCS, antibiotics and 5% IL-2 (Boehringer). Cultures were replenished twice weekly with fresh medium containing IL-2 and once with 5×105/ml donor PBMC. Virus growth was monitored weekly using a commercial p24 antigen assay (Coulter). Antigen positive cultures were expanded and cultured for a further 2 weeks to obtain 40 mls of virus containing supernatant which was stored at −70° C. until use. The results of the isolation of the viruses from the commercial sex workers is also shown in Table 1 below.
Viral Phenotypes
Virus-containing supernatant was used to assess the biological phenotype of viral isolates on MT-2 and co-receptor transfected cell lines. For the MT-2 assay, 500 ul of supernatant was incubated with 5×104 MT-2 cells in PRMI plus 10% FCS and antibiotics. Cultures were monitored daily for syncitia formation over 6 days. U87.CD4 cell expressing either the CCR5 or CXCR4 co-receptor were grown in DMEM with 10% FCS, antibiotics, 500 ug/ml G418 and 1 ug/ml puromycin. GHOST cells expressing minor co-receptors were grown in DMEM with 10% FCS, 500 ug/ml G418, 1 ug/ml puromycin and 100 ug/ml hygromycin. Cell lines were passaged twice weekly by trypsination. Co-receptor assays were done in 12 well plates; 5×104 cells were plated in each well and allowed to adhere overnight. The following day 500 ul of virus containing supernatant was added and incubated overnight to allow viral attachment and infection and washed three times the following day. Cultures were monitored on days 4, 8 and 12 for syncitia formation and p24 antigen production. Cultures that showed evidence of syncitia and increasing concentrations of p24 antigen were considered positive for viral growth. The results of co-receptor usage of the viruses from the commercial sex workers is also shown in Table 1.
*date from November 1998
Sequencing
RNA was isolated from plasma and the gene fragments were amplified from RNA using reverse transcriptase to generate a cDNA followed by PCR to generate amplified DNA segments. The positions of the PCR primers are as follows, with the second of each primer pair being used as the reverse transcriptase primer in the cDNA synthesis step (numbering using the HIV-1 HXBr sequence): gag1 (790-813, 1282-1303), gag2 (1232-1253, 1797-1820), pol1 (2546-2573, 3012-3041), pol2(2932-2957, 3492-3515), enyl (6815-6838, 7322-7349), env2 (7626-7653, 7963-7986). The amplified DNA fragments were purified using the QIAQUICK PCR Purification Kit (Qiagen, Germany). The DNA fragments were then sequenced using the upstream PCR primers as sequencing primers. Sequencing was done using the Sanger dideoxyterminator strategy with fluorescent dyes attached to the dideoxynucleotides. The sequence determination was made by electrophoresis using an ABI 377 Sequencer. A mapped illustration of an HIV-1 proviral genome showing the pol, gag and env regions sequenced as described above, is shown in
Genotypic Characterization
To select the vaccine isolate or isolates, a survey covering portions of the three major HIV genes gag (313 contiguous codons, 939 bases), pol (278 contiguous codons, 834 bases) and env (229 codons in two noncontigous segments, 687 bases) was done (
Of a total of 31 isolates, 14 were from the Durban cohort (DU), 15 were from Johannesburg (GG and RB) and 2 from Cape Town (CT). Of these 30 were sequenced in the gag region, 26 in the pol region and 27 in the env region. The isolates that were sequenced are shown in Table 2.
The nucleic acid sequences from the Durban (DU) Johannesburg (GG, RB) and Cape Town (CT) cohorts were phylogenetically compared to all available published subtype C sequences (obtained from the Los Alamos HIV Sequence Database) including sequences from the other southern African countries and the overall subtype C consensus from the Los Alamos HIV sequence database. This comparison was done to ensure that the selected vaccine isolates were not phylogenetic outliers when compared to the Southern African sequences and the results of the comparison are shown in
Determination of a Consensus Sequence
Amino acid sequences were derived from the sequences shown in Table 2 and were used to determine a South African consensus sequence. The most frequently appearing amino acid at each position was selected as the consensus amino acid at that position. In this way, the consensus sequence was determined along the linear length of each of the sequenced gene fragments (gag, pol and env gene fragments). The alignments were done using the Genetics Computer Group (GCG) programs (Pileup and Pretty), which generates a consensus sequence in this manner. These resulted in the consensus sequence for each gene region. The alignments of the amino acid sequences and the resulting consensus sequences are shown in
The phylogenetic tree of amino acids showing a comparison of the South African sequences is set out in
The final choice of which isolate or isolates to use was based on the similarity of the sequence of the gag, env and pol genes of a particular isolate to the South African consensus sequence which had been derived as set out above as well as the availability of an R5 isolate which had good replication kinetics as shown in Table 1.
Selection of Vaccine Isolates
Based on the considerations and methodology set out above, three strains were selected from the acute infection cohort as the vaccine strains. The first strain is Du422 for the gag gene, the second strain is Du151 for the pol and env genes and the third strain is Du179 which is a possible alternative for the env gene. These three strains were selected for the following reasons.
(1) At the time the samples were obtained, Du 151 had been infected for 6 weeks and had a CD4 count of 367 cells per ul of blood and a viral load above 500,000 copies per ml of plasma. Given the high viral load, and the recorded time from infection, it is probable that the individual was still in the initial stages of viraemia prior to control of HIV replication by the immune system.
(2) At the time the samples were obtained, Du422 had been infected for 4 months with a CD4 count of 397 cells per ul of blood and a viral load of 17,118 copies per ml of plasma. In contrast to Du151 this individual had already brought viral replication under control to a certain extent.
(3) At the time the samples were obtained, Du179 had been infected for 21 months with a CD4 count of 394 cells per ul of blood and a viral load of 1,359 copies per ml of plasma.
Based on the analysis of the phylogenetic tree shown in
The pol sequences showed the highest values for the pairwise comparisons. Based on the analysis of the phylogenetic tree shown in
The env gene showed the greatest sequence diversity. Based on the analysis of the phylogenetic tree shown in
The clones representing the full length gene for each of the above viral genes were generated by PCR. Viral DNA present in cells infected with the individual isolates were used for the pol and env clones, and DNA derived directly from plasma by RT-PCR was used for the gag clone. Total DNA was extracted from the infected cell pellets using the QIAGEN DNeasy Tissue Kit. This DNA was used in PCR reactions using the following primers (HXBR numbering, Los Alamos database) in a nested PCR amplification strategy:
gag: outer,623-640, and 2391-2408; inner, 789-810 and 2330-2350;
pol: outer,2050-2073, and 5119-5148; inner,2085-2108, and 5068-5094;
env: outer, 6195-6218, and 8807-8830; inner, 6225-6245, and 8758-8795.
The PCR products were blunt-end cloned into pT7Blue using the Novagen pT7Blue Blunt Kit. The inserts were characterized by doing colony PCR to identify clones with gene inserts. The identity of the insert was confirmed by sequencing the insert on both strands and comparing this sequence to the original sequence.
Modification of Clones
Several modifications were introduced to the cloned genes, as shown in SEQ ID NOs: 7-12. In order to increase levels of expression of proteins, the DNA sequence was resynthesized and the following modifications were made:
the codon usage was changed to reflect human codon usage for increased expression; and
the inhibitory and rev responsive elements were also removed.
The modifications to the gag gene sequence of Du422 are shown in SEQ ID NOs: 7 and 8.
Also for the DNA, modified vaccinia ankara (MVA) and BCG vaccines, the pol gene was truncated so that only the protease, reverse transcriptase and RNAse H regions of the pol gene will be expressed. In addition, the active site amino acid motive YMDD has been mutated to YMM so that the expressed reverse transcriptase will be catalytically inactive. The modifications to the pol gene of Du151 are shown in SEQ ID NOs: 9 and 10.
Synthetic Genes
The complete gag and env genes were resynthesized to optimize the codons for expression in human cells, also shown in SEQ ID NOs: 9 to 12. During this process the inhibitory sequences (INS) and rev responsive elements (RRE) are removed which has reported to result in increased expression. The gag gene myristylation signal was mutated as described above and as shown in SEQ ID NOs: 7 and 8.
The following material has been deposited with the European Collection of Cell Cultures, Centre for Applied Microbiology and Research, Salisbury, Wiltshire SP4 OJG, United Kingdom (ECACC).
The deposit was made under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure and regulations thereunder (Budapest Treaty).
This application is a continuation of U.S. patent application Ser. No. 10/332,413, filed Sep. 12, 2003. The disclosure of U.S. patent application Ser. No. 10/332,413 is incorporated in its entirety herein.
| Number | Date | Country | |
|---|---|---|---|
| 60216995 | Jul 2000 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 10332413 | Sep 2003 | US |
| Child | 11724551 | Mar 2007 | US |
