WO1999000490A2

WO1999000490A2 - Attenuated human immunodeficiency virus vaccine

Info

Publication number: WO1999000490A2
Application number: PCT/CA1998/000617
Authority: WO
Inventors: Mark A. Wainberg
Original assignee: The Sir Mortimer B. Davis-Jewish General Hospital
Priority date: 1997-06-26
Filing date: 1998-06-26
Publication date: 1999-01-07
Also published as: WO1999000490A3; CA2208946A1; AU8096998A

Abstract

Reverse transcription of retroviruses is initiated from an 18 nucleotide primer binding site (PBS), located within the 5' region of viral genomic RNA, to which the host cell-derived tRNA primer is annealed and also involves viral genomic sequences outside the PBS. Significantly lower levels of viral DNA are detected in cells infected with proviral DNA clones of human immunodeficiency virus (HIV) selectively deleted in regard to a segment found immediately downstream of the PBS. This segment is involved in efficient expression of each of viral DNA, mRNA, and infectious virus. The deleted DNA clones are useful for the preparation of an HIV vaccine.

Description

Attenuated Human Immunodeficiency Virus Vaccine

Field of the Invention

The invention relates to restriction of replication of human immunodeficiency virus (HIV) . More particularly, the invention relates to means of affecting efficiency of expression of viral DNA, mRNA and infectious virus. The invention relates to an attenuated virus vaccine based on DNA clones of HIV selectively deleted in regard to a segment found immediately downstream of the primer binding sequence (PBS) .

Background of the Invention

Attenuated viruses can be used to develop live-virus vaccines. Attenuated virus can replicate to a limited extent or not at all, yet can successfully immunize and thereby protect against subsequent exposure to a live and virulent form of the virus. One method of obtaining attenuated viruses is to genetically modify the viral genome.

It is of great interest to develop a vaccine against the retrovirus, human immunodeficiency virus (HIV) . Retroviruses have a single (+) strand RNA genome. After infection, this genome is transcribed by an enzyme called reverse transcriptase into (-) single-stranded DNA from which the complementary DNA strand is made, resulting in (±) double- stranded proviral DNA. The proviral DNA is incorporated into the chromosomes of host cells, from which viral mRNA may then be transcribed.

Reverse transcription begins at the primer binding sequence (PBS) of unspliced retroviral RNA, to which a tRNA primer is positioned (38) . The PBS of human immunodeficiency virus type 1 (HIV-1) is located approximately 180 nucleotides (nt) from the 5' terminus of genomic RNA and is flanked at its 5' end by a region referred to as R/U5 (49) . This R/U5 region possesses a number of functional activities, including a role in packaging of viral RNA, binding of the Tat transactivator protein, and involvement in reverse transcription and integration of proviral DNA (1, 7, 12, 13, 21, 24, 25, 28, 34, 44, 52, 56, 57). A 133-nt noncoding/untranslated region is located downstream of the PBS and upstream of the gag initiation codon (49) . The function of this sequence, especially its 5' portion, is not well understood, although its 3' end is thought to be involved in packaging, splicing and dimerization of genomic RNA, and translation of viral proteins (2, 9, 11, 15, 32, 35, 41, 42, 45, 51) .

The PBS region of HIV-1 RNA and surrounding sequences appear to be highly structured as determined by computer modelling and chemical analysis (6, 8, 22) . The unfolding of the tRNA primer and of the RNA template is thought to be mediated by the viral nucleocapsid protein (NCp) (30, 31, 37) . Formation of the reverse transcription initiation complex involves base pairing between the PBS and a complementary 18-nt region at the 3' end of tRNA, as well as additional interactions between sequences that neighbour the PBS and the remainder of the tRNA primer. In avian retroviruses, the efficiency of a tRNA^Trp-PBS complex in initiation of reverse transcription was enhanced by inclusion of viral genomic sequences upstream of the PBS and the T C loop of tRNA^Trp (1, 34). Furthermore, disruption of a stem-loop structure, i.e. the U-IR stem near the PBS, caused diminished reverse transcription in both avian and murine retroviruses (12, 13, 44, 48) .

Summary of the Invention

Applicant has studied the role in viral replication of non-coding sequences that lie downstream of the PBS, by introducing deletions immediately downstream of this region. Applicant has shown that deletion of the 7-nt segment (pHIV/del-7) has relatively minor effects on in vivo reverse transcription of viral DNA product in MT-4 cells; whereas a 54- nt deletion (pHIV/del-LD) significantly reduced such transcription. Applicant also has shown that this 54-nt sequence is independently involved in efficient expression of viral mRNA; cells transfected with the pHIV/del-LD mutant expressed significantly lower levels of viral mRNA compared with wild type or pHIV/del-7 viruses. The pHIV/del-7 virus displayed similar replication kinetics to those of wild-type viruses, while the pHIV/del-LD virus, as well as viruses containing other deletions in this region, were significantly impaired in this regard.

This invention describes deletions in the HIV genome that result in restricted virus replication. These deletions occur within a 133-nt sequence located downstream of PBS and upstream of the gag initiation codon. The deletions include all or part of the hereinafter described 54-nt sequence.

This invention relates to an attenuated virus which contains such a deletion. Such virus can be used as a vaccine for protection against HIV and in the prevention of acquired immunodeficiency syndrome (AIDS) . In one aspect, this invention provides a nucleic acid encoding a mutant human immunodeficiency virus containing a deletion in the wild type sequence, wherein the deletion is found immediately downstream of the primer binding sequence, said deletion being effective to attenuate the virus expressed by the primer binding sequence.

In another aspect, this invention provides an expressible genetic construct containing the nucleic acid as described above for transforming cells.

In a further aspect, this invention provides an attenuated human immunodeficiency virus containing the nucleic acid as described above.

In another aspect, this invention provides a method for making an attenuated human immunodeficiency virus, which method comprises: making a proviral DNA clone of human immunodeficiency virus comprising the nucleic acid as described above; using the proviral DNA clone to transfect cells; and harvesting the attenuated human immunodeficiency virus from the transfected cells. In a further aspect, this invention provides a vaccine which comprises an attenuated human immunodeficiency virus as described above and a pharmaceutically acceptable carrier or diluent.

In a further aspect, this invention provides a method of immunizing a mammal against human immunodeficiency virus which comprises administering to the mammal an effective immunizing dose of the vaccine described above.

Brief Description of the Drawings

These and other objects, features, and many of the attendant advantages of the invention will be better understood upon a reading of the following detailed description of the invention when considered with the accompanying drawings herein.

Fig. 1: Schematic depiction of deletion mutations surrounding the PBS of HIV-1 proviral DNA. pHIV/del-7 represents a 7-nt deletion immediately downstream of the PBS; pHIV/del-LD represents a 54-nt-deletion also immediately downstream of the PBS and containing the aforementioned 7-nt sequence. The initiation codon of the gag gene is indicated along with relevant nucleotide positions.

Fig. 2: Viral replication capacity of various constructs. Cell-free viruses harvested from COS-7 cells transfected with various molecular constructs (72 hr post-transfection) were used to infect MT-4 cells. Culture fluids were collected and monitored for reverse transcriptase activity. Decreased viral production in MT-4 cultures after 1 week in the case of cells infected by pHIV/ T (D) and pHIV/del-7 (O) ) was due to viral cytopathology; fresh cells were not added to these cultures. (o) designates infection by pHIV/del-LD virus while (Δ) represents mock infected cells. Fig. 3: Relative quantities of viral RNA packaged into viral structures. COS-7 cells were transfected with either pHIV/del-LD (grey) or pHIV/WT (dotted) . After 60 hr, viruses in culture fluids were purified by sucrose gradient ultracentrifugation. RNA was extracted from viruses equalized on the basis of p24 content and quantified by slot blot and liquid scintillation analysis. Experiments were performed using 3 replicate samples; error bars represent standard deviation. In some cases, viral RNA was digested with_RNase and all hybridizable material was eliminated. Results are standardized to 100 for pHIV/WT (7ng) .

Fig. 4: Detection of viral DNA. Viruses harvested from culture fluids of COS-7 cells, that had been transfected with various molecular constructs, were standardized on the basis of p24 content and used to infect MT-4 cells. Total cellular DNA (approximately 50 μg) was isolated from infected cells at 4 and 8 hr after infection, and subjected to PCR analysis using primers that specifically amplify minus-strand strong-stop DNA (59) . Primers amplifying -globin were used as an internal control to monitor the input of sample DNA (59) . Mock infections involved culture fluids derived from COS-7 cells that had been transfected with DNA from cells inoculated with heat-inactivated viruses. Lanes 1-3: cells exposed to heat-inactivated viruses HIV/ T, HIV/del-7, and HIV/del-LD, respectively; lanes 4, 6, 8: cells infected with HIV/WT, HIV/del-7, and HIV/del-LD, respectively; lanes 5, 7, 9: cells infected with HIV/WT, HIV/del-7, and HIV/del-LD in the presence of 2μM AZT. In the case of lanes 1-9, cells were maintained for 4 hr after exposure to virus prior to extraction of DNA. Lanes 10-15: same order of experiments as lanes 4-9 except that DNA was extracted after 8 hr. Lanes 16-19: several dilutions of HxB2D plasmid as a positive control (i.e. 10-fold dilutions of plasmids in terms of copy numbers, i.e., 5 x 10²; 5 x 10³; 5 x 10⁴; and 5 x 10⁵) .

A. Detection of minus-strand strong-stop DNA.

B. Detection of viral DNA generated after the first template switch. C. Detection of viral DNA generated after the second template switch.

D. PCR amplification of β-globin DNA as an internal control. Fig. 5: Detection of minus-strand strong-stop DNA.

Infection by pHIV/del-LDl , lanes 1,5; pHIV/del-LD2 , lanes 2,6; pHIV/del-LD3, lanes 3,7; pHIV/WT, lanes 4,8; inoculation of cells with heat-inactivated wild-type virus, lane 9. Positive controls of serially diluted HXB2D plasmids are as shown in Fig. 4.

Fig. 6A: Northern blots for detection of viral RNA. Total cellular RNA was purified from COS-7 cells 16 hr after transfection with either pHIV/del-LD or pHIV/WT. Lane 1: RNA (20μg) from cells transfected with pHIV/del-LD; lane 2: RNA (20μg) from cells transfected with pHIV/WT; lane 3: RNA (10 μg) from cells transfected with pHIV/del-LD; lane 4: RNA (lOμg) from cells transfected with pHIV/WT; lane 5: RNA (20μg) from mock-transfected COS cells. Molecular size markers are indicated.

Fig. 6B: Quantitative determination of viral RNA transcripts by slot blot. Total cellular RNA was harvested from COS-7 cells and purified at 16, 24, 48, and 72 hr, respectively, after transfection with various molecular constructs. Relative intensities were calculated by comparison with levels of radioactivity obtained with wild-type transfections after 72 hr, defined as 100, (i.e. 2478 cpm) . Standard deviations (SD) are indicated by error bar (four separate experiments) . Fig. 7: RNA stability assay. Actinomycin-D was added to culture medium at 36 hours after transfection of COS-7 cells and total cellular RNA was extracted at 0, 1, 3, and 6 hours thereafter. Levels of viral RNA were determined by RT-PCR (top of Figure) and analysed by molecular imaging (bottom) . "Mock" designates a RT-PCR reaction performed with wild-type HIV RNA in the absence of reverse transcriptase. HIV/del-LD and HIV/WT designate infections performed with HIV/del-LD and wild-type constructs, respectively.

Fig. 8: Viral protein analysis by Western blot. Proteins isolated from COS-7 cells were analysed by Western blot as described in the description: proteins from COS cells transfected with pHIV/del-LD (lane 1) , from COS cells transfected with pHIV/WT (lane 2) ; positive control, using proteins derived from MT-4 cells infected by HIV-IIIB (lane 3) . Proteins from mock-transfected COS-7 cells (lane 4) . Detailed Description of the Invention

A 54-nt segment in the non-coding region of the HIV-1 genome, downstream of the PBS, is important for viral replication in each of two principal ways. First, at least part of this region is necessary for efficient reverse transcription of viral DNA product, including that which is generated both prior to, as well as after, each of the two template switch events. In addition, at least part of this region is important for the efficient generation of viral mRNA and consequently for the synthesis of viral protein and infectivity.

Each of these effects (i.e. on reverse transcription and on synthesis of viral transcripts) seems to be independent of the other. Applicant has shown this through: (a) studies in which infection by viruses containing relevant deletions in viral RNA yielded extremely low levels of viral DNA products generated both before and after template switching, and (b) independent experiments in which transfection of cells using deleted DNA constructs failed to generate significant levels of viral mRNA.

Applicant has further shown in related work that long term culture of mutated viruses in cells, yielded revertant viruses that possess infectivities similar to that of the wild type. The reason for such reversion is one or more compensatory mutations. HIV-1 has a highly error-prone reverse transcriptase, a high rate of replication, and lacks 3' to 5' proofreading activity. Applicant and others have shown that the fidelity of DNA-dependent DNA polymerization of M184V- mutated HIV-1 RT is significantly higher than that of wild-type RT (61) . Applicant further demonstrated that the fidelity of RNA-dependent DNA polymerization was also significantly higher than that of wild-type RT (61) .

Applicant has designed attenuated viruses which incorporate deletions in the region immediately downstream from PBS. Further, in one aspect of the invention, a second mutation is incorporated into the attenuated virus, such as M184V, to diminish the rate of spontaneous mutation in the attenuated virus through successive generations, thus reducing the chance of revertant mutations. Replication of virus deletion mutants The mutations introduced into proviral DNA constructs

(Fig. 1) include a deletion of the conserved 7-nt stretch located immediately downstream of the PBS (pHIV/del-7) , and an extensive 54-nt deletion downstream of the PBS containing the aforementioned 7-nt segment (pHIV/del-LD) (Fig. 1) . In addition, the 54-nt deletion region was subdivided by smaller deletions termed pHIV/del-LDl, pHIV/del-LD2, and pHIV/del-LD3 (Fig. 1) .

To investigate the replication potential of these constructs, viruses (containing 50 ng p24) derived from COS-7 cells, that had been appropriately transfected, were used to infect MT-4 cells. RT and p24 are two viral proteins. The levels of these proteins which are produced by the infected cells is a measure of the ability of the virus to infect the cells. Fig. 2 shows that wild-type virus (pHIV/WT) and one of the deletion mutants (pHIV/del-7) replicated efficiently, as determined by levels of RT activity in culture fluids after 3 and 7 days. In contrast, the pHIV/del-LD mutant was significantly impaired in ability to produce viral progeny (Fig. 2) . Further analysis revealed that the pHIV/del-LD3 mutant was most severely diminished in its ability to replicate.

Applicant also studied the ability of viruses derived from transfections of COS-7 cells to infect MT-4 cells, using a p24 antigen capture assay. In this instance, viruses were also examined that had been subjected to more extensive deletion mutagenesis than that found in pHIV/del-LD. Two different concentrations of viral inoculum were used in each case. The results of Table 1 show that the pHIV/del-7 construct yielded similar levels of p24 to wild-type virus over 13 days, while both pHIV/del-LD and the pHIV/delLD-3 construct, with a deletion of 16-nt at the 3' end of the 54-nt LD deletion, were severely impaired and produced only low levels of p24 for at least 90 days in culture. In contrast, little or no effect was observed when pHIV/del-LDI, deleted of 18 nt at the start of this 54-nt segment, was employed. Moderate inhibition of p24 synthesis was noted when pHIV/LD-2 was studied; the latter construct lacks a stretch of 20 nt at the centre of this 54-nt region. These findings are consistent with the data of Fig. 2 and the work presented below on synthesis of viral DNA in infected cells. TABLE 1. Levels of p24 antigen expression in infected MT-4 cells³

Viral construct Inoculum p24 concn (ng/ml) on days (ng of p24)

7 10 13 30 90 pHIV/WT 50 23.5 27.3 19.7^b 10 18.9 16.8 13.9^b pHIV/del-7 50 15.6 19.4 16.5^b 10 12.7 21.9 17.9^b pHIV/del-LD 50 1.5 2.0 2.4 2.0 1.8 10 0.4 1.1 1.3 1.2 1.3 pHIV/del-LDl 50 18.7 26.2 17.4^b 10 18.5 20.8 16.8^b pHIV/del-LD2 50 10.3 14.5 15.7^b 10 8.6 11.4 13.2^b pHIV/del-LD3 50 0.9 1.2 2.1 1.2 1.5 10 0.6 1.6 1.9 1.6 1.9 ^a MT-4 cells were infected with various viral constructs, and p24 levels in culture fluids were measured. ^b After day 13, cytotoxicity resulted in the death of cultures that produced relatively high levels of p24. In contrast, cultures infected by the pHIV/del-LD and pHIV/del-LD3 viruses continued to generate low levels of p24 activity over extensive periods.

Production of minus-strand strong-stop DNA in infected cells

Applicant found that similar levels of viral RNA were packaged into viruses derived from COS-7 cells that had been transfected 72 hr earlier with its various constructs (results are shown for pHIV/WT and pHIV/del-LD based on RNA:p24 ratios)

(Fig 3) . When these RNA preparations were digested with RNase as a negative control, little or no hybridizable material remained, indicating that contaminating viral DNA was not present in these preparations.

Since previous work had shown that certain sequences surrounding the PBS were involved in reverse transcription in cell-free systems (24, 25, 33), applicant investigated whether the modifications introduced into its constructs would result in impaired generation of viral DNA. Toward this end, total cellular DNA was isolated at 4 and 8 hrs after infection of MT-4 cells with viruses derived from COS-7 cells, and analysed by PCR, using primer pairs that specifically amplify minus- strand strong-stop DNA as well as viral DNA that is generated after each of the first and second template switch events.

Applicant found that similar levels of minus-strand strong-stop DNA were present in MT-4 cells infected by each of pHIV/del-7 (Fig. 4A, lanes 6 and 12) and wild-type virus (Fig. 4A, lanes 4 and 10) after 4 and 8 hr. In contrast, MT-4 cells infected with the pHIV/del-LD mutant contained significantly decreased levels of minus-strand strong-stop DNA (Fig. 4A, lanes 8 and 14) (i.e. about 10 times less than with wild-type virus as quantified by densitometry) . As a control, applicant performed mock infections using culture fluids of COS-7 cells that had been transfected with DNA from cells inoculated with heat -inactivated viruses and was unable to detect a DNA signal (Fig. 3A, lanes 1, 2, 3 for wild-type, pHIV/del-7 and pHIV/del-LD, respectively. Consistent results were obtained with total cellular DNA using primer pairs that amplify DNA that is present after the first template switch (Fig. 4B) as well as full-length reverse transcribed DNA (Fig. 4C) (60) . As an additional important control, applicant treated cells with 2 μM AZT in order to prevent synthesis of viral DNA product generated after the first template switch. Indeed, applicant found, as expected, that such treatment did not affect levels of minus-strand strong-stop DNA in the case of either wild-type virus (lanes 5 and 11) or pHIV/del-7 (lanes 7 and 13) (Fig. 4A) . Nor, in fact, did the presence of AZT affect the already diminished levels of minus-strand strong-stop DNA found in cells infected by pHIV/del-LD_ (lanes 9 and 15) (Fig. 4A) .

In contrast, treatment with 2 μM AZT significantly impaired synthesis of DNA products generated after both the first and second template switch events for each of the viruses tested (Figs. 4B,C) (compare lane 4 with 5, lane 6 with 7, 8 with 9, 10 with 11, 12 with 13, 14 with 15) . In the case of full-length product (Fig. 4C) , it should be noted that pHIV/del-LD, as expected, yielded a smaller DNA product (lanes 8 and 14) than that obtained with wild-type virus. In this case, treatment with AZT prevented the appearance of any detectable DNA product (lanes 9 and 15) .

Thus, at least part of the 54-nt untranslated sequence, located immediately downstream of the PBS, is necessary for both efficient reverse transcription and infectivity. As expected from the results of Fig 4, far less proviral DNA became integrated into MT-4 cells after infection with pHIV/del-LD than with pHIV/WT or pHIV/del- 7, but persisted for up to 3 months. No evidence of revertant virus was observed as determined by sequencing during extensive cultivation, although p24 antigen could be detected at low levels for as long as 3 months.

To further define minimal necessary sequences within this 54-nt region, the pHIV/del-LDl, pHIV/del-LD2 , and pHIV/del-LD3 viruses were used to infect MT-4 cells and levels of reverse transcribed DNA were determined (Fig. 5) . Molecular imaging analysis showed, in comparison with wild-type virus, (lanes 4, 8), that pHIV/del-LD3 was severely impaired (i.e. >90%) in synthesis of minus-strand strong-stop DNA (lanes 3, 7) . Only a modest diminution (=50%) in generation of such material occurred when pHIV/del-LD2 was studied (lanes 2, 6) while no effect whatever was seen in the case of pHIV/del-LDl (lanes 1, 5) . These findings are consistent with the results described above on viral replication. Role of the untranslated region downstream of the PBS on viral gene expression

The above data indicate that the 54-nt region is involved in generation of viral DNA, consistent with observations in cell-free systems (37) . However, the observed reductions (=10-fold) might not have led directly to the near-lethality of pHIV/del-LD since post-integrational effects, e.g. generation of viral mRNA and proteins, might also have played a role. Applicant therefore assessed what role the untranslated region downstream of the PBS might play in expression of viral mRNA. Fig. 6 depicts the results of Northern blot analysis of viral RNA extracted from COS-7 cells transfected with either mutant or wild type constructs.

Levels of viral RNA transcripts in cells transfected with pHIV/del-LD were much lower than those in cells transfected with pHIV/WT, although the major three bands representing unspliced, singly spliced and multiply spliced RNA were present in each case (Fig. 6A) . Similar viral RNA transcript patterns were observed in COS-7 cells transfected with pHIV/del-7 and wild type virus.

These results were further confirmed by quantitative slot blot analysis. Fig. 6B shows that dramatically reduced levels of RNA transcript were present in cells transfected with pHIV/del-LD compared with wild type virus (pHIV/WT) or pHIV/del-7. Differences were most pronounced at early time points after transfection (16 hr) .

To exclude the possibility that the reduced levels of viral mRNA in pHIV/del-LD transfected cells were due to instability, applicant determined the half-lives of the viral mRNA molecules produced following transfection of COS-7 cells by wild-type and mutated constructs. Toward this end, cells were treated with actinomycin D at 36 hr after transfection, as described in the Examples, and total RNA was extracted at 0,1, 3 and 6 hr thereafter and reverse transcribed to yield DNA. The results of specific PCR amplifications revealed an expected disappearance of relevant amplified genetic material over time (top of Fig. 7) . Consistent with the results of Fig. 6, cells transfected with the HIV/del-LD construct produced much lower overall levels of mRNA than did those transfected by wild-type material. However, the rates of disappearance of viral RNA in both cases were nearly identical as shown by molecular imaging analysis (bottom of Fig. 7) . Indeed, no differences in regard to stability were observed among mRNA molecules derived from the wild-type, pHIV/del-LD, pHIV/del-LDl, pHIV/del-LD2 , or pHIV/del-LD3 constructs. Effects on viral protein synthesis Applicant next investigated protein expression and viral assembly in COS-7 cells that had been transfected with wild-type DNA and the pHIV/del-LD construct. Toward this end, p24 detection and Western blot analyses were performed on culture fluids and cell lysates. As expected on the basis of the RNA transcript results described above, COS-7 cells transfected with pHIV/del-LD produced lower levels of both intracellular and extracellular p24 after 16 hr than cells transfected with pHIV/WT (Table 2) . Interestingly, transfection with pHIV/del-LD did not result in excess accumulation of intracellular p24 relative to other transfections, suggesting that viral protein assembly had proceeded normally.

TABLE 2. Intracellular and extracellular p24 levels in COS-7 cells transfected with pHIV/del-LD or pHIV/WT

p24 level (ng/ml)^a h after Intracellular Extracellular transfection pHIV/del-LD pHIV/WT pHIV/del-LD pHIV/WT transfection transfection transfection transfection

16 5.22 ± 0.48 170 ± 15.2 4.49 ± 0.32 187 ± 16.8 24 17.9 ± 1.55 241 ± 22.2 18.7 ± 16.2 258 ± 25.3

48 64.8 ± 6.02 233 ± 24.5 97.0 ± 10.2 304 ± 29.8

72 62.9 ± 5.89 245 ± 23.3 145 ± 13.5 300 ± 31.2

^a At various times after transfection, both intracellular and extracellular viral p24 levels were determined. Data are means + standard deviations from four separate experiments. It was also important to determine whether the diminished synthesis of viral proteins, associated with pHIV/del-LD, would affect the profiles of the viral proteins produced by transfected COS-7 cells, using a system in which the same total amount of protein was analyzed in each case by gel electrophoresis. Fig. 8 is a Western blot analysis of proteins produced by COS-7 cells that had been transfected by pHIV/del-LD (lane 1), pHIV/WT (lane 2), or mock-transfected (lane 4) . Lane 3 represents MT-4 cells infected by wild-type HIV. Each of lanes 1-3 was equalized on the basis of amount of p24 as determined by ELISA assay.

Applicant found that viral protein profiles were essentially nondistinguishable among COS-7 cells transfected by the mutant (Fig. 8, lane 1) or by the wild-type construct (lane 2) or MT-4 cells infected by wild-type virus (lane 3) . Nor were differences observed in regard to transfections by the pHIV/del-LDl, pHIV/del-LD2 or pHIV/del-LD3 constructs. Thus, deletion of the 54-nt stretch downstream of the PBS did not affect patterns of viral protein synthesis but rather led to a marked decrease in levels of all viral proteins produced. This is because the untranslated sequences downstream of the PBS can influence the production of infectious progeny virus by affecting both reverse transcription and the expression of viral mRNA. Revertant Viruses

In a related system, applicant studied revertant viruses. The dimerization initiation site (DIS) , downstream of the long terminal repeat within the human immunodeficiency virus type 1 (HIV-1) genome, can form a stem-loop structure (SLl) that has been shown to be involved in the packaging of viral RNA. In order to further determine the role of this region in the virus life cycle, applicant deleted the 16 nucleotides (nt) at positions +238 to +253 within SLl to generate a construct termed BH10-LD3 and showed that this virus was impaired in viral RNA packaging, viral gene expression, and viral replication. Long-term culture of these mutated viruses in MT-2 cells, i.e., 18 passages, yielded revertant viruses that possessed infectivities similar to that of the wild type. Cloning and sequencing showed that these viruses retained the original 16-nt deletion but possessed two additional point mutations, which were located within the p2 and NC regions of the gag coding region, respectively, and which were therefore named MP2 and MNC. Site-directed mutagenesis studies revealed that both of these point mutations were necessary to compensate for the 16-nt deletion in BH10-LD3. A construct with both the 16-nt deletion and the MP2 mutation, i.e., LD3-MP2, produced approximately five times more viral protein than BH10-LD3 while the MNC mutation, i.e., construct LD3-MNC, reversed the defects in viral RNA packaging. Applicant also deleted nt +261 to +274 within the 3' end of SLl and showed that the diminished infectivity of the mutated virus, termed BH10-LD4, could also be restored by the MP2 and MNC point mutations. Therefore, compensatory mutations within the p2 and NC proteins, distal from deletions within the DIS region of the HIV genome, can restore HIV replication, viral gene expression, and viral RNA packaging to control levels. This work indicates that a live attenuated HIV vaccine might revert to wild-type virulence through mutations. It also indicates that a 16 nt deletion may be of insufficient length in certain cases for use in an attenuated HIV vaccine.

Example 1 Molecular clones with deletion mutations in sequences surrounding the PBS

The HxB2D recombinant clone of infectious DNA, obtained from the NIH reagent repository, was used as a starting material for further genetic alteration. Applicant modified a previously described polymerase chain reaction (PCR) based mega-primer mutagenesis procedure to generate deletions in the vicinity of the PBS (47) .

The primer selected for the 7-nt deletion, (i.e. pHIV/del-7) , immediately downstream of the PBS (i.e. nt positions 654-660) , was

5' -TGGCGCCCGAACAGGGACCTGAAAGGGAAACCAGAG-3' . The primer for deletion of the 54-nt segment (i.e. pHIV/del-LD) , also downstream of the PBS, (i.e. nt positions 654-707) was

5' -TGGCGCCCGAACAGGGACCGCGCACGGCAAGAGGCG-3' . These were used as forward primers in conjunction with a backward primer (termed Pst 1, nt positions 1405-1422, 5' -CCATTCTGCAGCTTCCTC-3' ) to specifically amplify sequences in regard to each of these deletions. The resulting amplified products were used as mega primers with an additional primer, termed UPBS (upstream of primer binding site) located at the 5' terminus of the R region (5' -AGACCAGATCTGAGCCTGGGAG-3 ' ) .

Amplified fragments were then digested with Bgl II and Pst I and were inserted into a pSVK3 vector (Pharmacia Biotech, Montreal, Quebec, Canada). The cloned fragments were sequenced to verify that correct modifications of viral gene sequences had been made and were inserted into the HXB2D clone of infectious DNA as described previously (36) .

To further define minimal sequences in the 54 -nt deletion (pHIV/del-LD) , three additional constructs were generated that deleted sequences at nt positions 654-671,

672-691, and 692-707, respectively. These were constructed using the following primers:

5' -GAGAGAGCTCTGGGTCCCTGTTCGGCG-3 ' ,

5 ' -CCGTGCGCGCTTCAGCAAGCCGAGTCTTTCCCTTTCGCTTTC-3 ' , and 5 ' -CCGTGCGCGCCTGCGTCGAGAGAGC-3 ' , in conjunction with the primer UPBS (see above) . Fig. 1 is a graphic description of the mutant viruses generated. Wild-type HXB2D viral DNA was designated pHIV/WT.

Example 2 Replication potential of viral constructs

Molecular constructs containing the above mutations in leader regions surrounding the PBS were purified twice by CsCl₂ gradient ultracentrifugation. These plasmids were transfected into COS-7 cells using a standard calcium co-precipitation procedure (40) . Virus-containing culture fluids were harvested approximately 72 hr after transfection and were clarified by centrifugation for 30 min at 4°C_at 3000 rpm, prior to filtration with a 0.2 μm-pore size sterile membrane. Viral preparations were stored at -70°C until use.

For purposes of infection, the viral stock was thawed and treated with 100 U DNase I in the presence of 10 mM MgCl₂ at 37°C for 1 hr to ensure that any contaminating plasmids had been eliminated from the transfection inocula (36) . Infection of MT-4 cells was performed by incubating cells at 37°C for 2 hr with virus (50 ng p24) , following which the cells were washed three times with PBS and incubated at 37°C with fresh medium. In some experiments, HIV-IIIB , kindly provided by Dr. R.C. Gallo, National Institutes of Health, Bethesda, MD, was used as a positive control . Culture fluids were monitored for virus production by means of reverse transcriptase assay (10) and by p24 (capsid protein, CA) antigen-detection enzyme-linked immunosorption assay (ELISA) (Abbott Laboratories, Abbott Park, IL) .

Example 3

Detection of viral DNA At various times after infection (4-8 hr) , MT-4 cells were collected and washed extensively with serum-free medium. To ensure that no contaminating plasmids were present, fluids from each wash were routinely checked by PCR using HIV-specific primers (36) . Total cellular DNA was then isolated from these cells (40) and analysed by PCR using specific primer pairs to amplify minus-strand strong-stop DNA (20, 60) . Cellular DNA isolated from cells inoculated with heat-inactivated wild-type viruses served as a negative control to ensure that potentially contaminating plasmids had been eliminated. For minus-strand strong-stop DNA, UPBS was employed as a forward primer, located at the 5' terminus of the ' R' region (nt 468-489) (49), while the backward primer was AA55' (nt 621-604) , modified from a previously published procedure (60) . The expected product of this primer pair (i.e. UPBS/AA55') is 153 bp in length.

To amplify viral DNA generated after the first template switch, applicant employed U3 as a forward primer (nt 1-21) and AR as a backward primer (nt 532-511) . To amplify viral DNA made after the second template switch, applicant used UPBS as a forward primer and PST, in the gag gene, as a backward primer (nt 1422-1398) . As a negative control, applicant also employed cells that had been pre-treated with 2 μM AZT for 3 hours prior to viral inoculation, and maintained these cells in the presence of drug for an additional 4-8 hr, prior to extraction of total DNA. PCR assays were performed with 50 μg of sample DNA, 50 mM Tris-Cl (pH 8.0), 50 mM KCl , 2.5 mM MgCl₂, 2.5 U Taq polymerase, 0.2 mM dNTPs, 10 pmols of ³²P-end-labelled forward primer, and 20 pmols of unlabelled backward primer. Reactions were standardized by simultaneous amplification of -globin sequences as an internal control (36, 60) and involved 30 cycles in which samples were subjected to 94°C (1 min), 60°C (1 min) and 72°C (1 min).

Example 4

Analysis of viral RNA by Northern/slot blot

Analysis of viral mRNA expression in COS-7 cells, transfected with various DNA constructs, was performed by slot and Northern blot procedures as described (10) . The efficiency of transfection was routinely monitored by detection of viral CA, using monoclonal anti-p24 antibodies in an immunofluorescence assay (10) . For Northern blots, total cellular RNA extracted from COS-7 cells was purified using a commercial RNA extraction kit (Biotecs, Houston, TX) .

The extracted RNA was treated with 100 U DNase I, followed by phenol-chloroform extraction and ethanol precipitation, to ensure removal of any contaminating plasmids and cellular DNA. The RNA pellets were resuspended in diethylpyrocarbonate-treated double-distilled water. RNA samples (up to 20 μg) were fractionated on 1% agarose gels containing formaldehyde as denaturant (10) . RNA molecules were transferred to a Hybond-N nylon membrane (Amersham, Toronto, Canada) and hybridized using pBHIO viral DNA as a radiolabelled probe (Nick translation system, Life Technologies, Toronto, Canada) as described (10) .

To quantify viral RNA transcripts derived from COS-7 cells, total cellular RNA (harvested at various times after transfection) was immobilized onto nylon membranes, using a slot blot apparatus, followed by UV irradiation (Amersham) . Hybridization reactions were performed as described for Northern blots (10) . The quantity of viral RNA was determined by counting relevant filter pads by liquid scintillation.

In some cases, viral RNA that had been packaged into virions (purified by sucrose gradient centrifugation) was also quantified by the slot blot protocol. To rule out the possibility that the samples tested also contained residual DNA, that might have been hybridized by the radiolabelled DNA probe, RNAase digestion of RNA extracted from virions was performed using RNase A (Boehringer-Mannheim, Montreal, Canada) at a final concentration of 10 μg/ml at 37°C for 30 min, following which phenol : chloroform extraction was performed.

Example 5

RNA stability assay Thirty-six hours after transfection, actinomycin D was added into culture medium to block the transcriptional activity of RNA polymerase II (19) . At different times, e.g. 0, 1, 3, and 6 hours after addition of drug, total cellular RNA was extracted using an Ultraspec™-II RNA isolation system (Biotecs, Houston, TX) , and was treated with 100 U RNase- free DNase I which was then removed by phenol -chloroform extraction. Two μg RNA were used in reverse transcription reactions, using

5 ' -TTTATTGAGGCTTAAGCAGTGGG-3' (n156 to n178 ) as an antisense primer in a total volume of 20 μl . One μl of product was then amplified in a 15 cycle-PCR using 5' -AGACCAGATCTGAGCCTGGGAG-3' (ntl4 to nt35) as a sense primer and the same antisense primer mentioned above to yield a 65 bp DNA fragment. Products were analysed on 5% polyacrylamide gels and further quantified by molecular imaging analysis. Example 6

Detection of viral proteins produced by transfected COS-7 cells

Expression of viral proteins in transfected COS-7 cells was determined using a commercial kit for detection of p24 CA antigen and by RT assay as described (10) . Both intracellular and extracellular CA levels were determined in order to shed light on the efficiency of viral assembly.

Viral proteins were also analysed by Western blot as described (10) . For this purpose, protein samples (standardized on the basis of viral p24) were fractionated on 12% SDS-polyacrylamide gels, and transferred to nitrocellulose filters (10) . The latter were then blocked with 5% skim milk/0.05% Tween-20/phosphate-buffered saline at 37°C for 2 hr, followed by exposure to sera obtained from HIV-1 seropositive individuals (10) . After extensive washing with 0.05% Tween-20/phosphate-buffered saline, ¹²⁵I-labelled goat anti-human IgG (ICN, Mississauga, Canada) was added for 1 hr at 37°C. The filters were then washed three times, dried, and exposed to Kodak Xomat film at -70°C.

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Claims

WE CLAIM :

1. A nucleic acid encoding a mutant human immunodeficiency virus containing a deletion in the wild type sequence, wherein the deletion is found immediately downstream of the primer binding sequence, said deletion being effective to attenuate the virus expressed by the primer binding sequence .

2. A nucleic acid according to claim 1 wherein the deletion is within or includes the 54 nucleotide segment immediately downstream of the primer binding sequence.

3. A nucleic acid according to claim 2 wherein the deletion is within or includes the 16 nucleotide segment at the 3' end of the said 54 nucleotide segment.

4. A nucleic acid according to claim 1, 2 or 3 which includes a M184V substitution.

5. A nucleic acid according to any one of claims 1 to 4 wherein the human immunodeficiency virus is HIV-1.

6. An expressible genetic construct containing the nucleic acid of any one of claims 1 to 5 for transforming cells.

7. An attenuated human immunodeficiency virus containing the nucleic acid according to any one of claims 1 to 5.

8. A method for making an attenuated human immunodeficiency virus, which method comprises: making a proviral DNA clone of human immunodeficiency virus comprising the nucleic acid according to any one of claims 1 to 5; using the proviral DNA clone to transfect cells; and harvesting the attenuated human immunodeficiency virus from the transfected cells.

9. A vaccine which comprises an attenuated human immunodeficiency virus according to claim 7 and a pharmaceutically acceptable carrier or diluent.

10. A method of immunizing a mammal against human immunodeficiency virus which comprises administering to the mammal an effective immunizing dose of the vaccine of claim 9.

11. A method according to claim 10 wherein the mammal is a human.