CA2208946A1

CA2208946A1 - Attenuated human immunodeficiency virus vaccine

Info

Publication number: CA2208946A1
Application number: CA002208946A
Authority: CA
Inventors: Mark A. Wainberg
Original assignee: Sir Mortimer B Davis Jewish General Hospita
Current assignee: Sir Mortimer B Davis Jewish General Hospita
Priority date: 1997-06-26
Filing date: 1997-06-26
Publication date: 1998-12-26
Also published as: WO1999000490A2; WO1999000490A3; AU8096998A

Abstract

Reverse transcription of retroviruses is initiated from a 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 were 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 results in restriction of replication of human immunodeficiency virus (HIV). More particularly, the invention relates to controlling 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 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, even though 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 each of the tRNA primer and 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 tRNATrP-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 tRNATrP
(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).
Applicant has studied the role in viral replication of non-coding sequences that lie downstream of the PBS, by introducing a deletion of seven nt immediately downstream of this region (33), designated pHIV/del-7. Applicant has also generated a 54-nt deletion of the 5' portion of the noncoding region, designated pHIV/del-LD, located immediately downstream of the PBS and containing the aforementioned 7-nt sequence.
Applicant's previous studies have shown that the first of these deletions is not required for cell-free synthesis of - - -minus-strand strong-stop DNA in reactions performed with recombinant RT (reverse transcriptase), NCp, tRNALYs3, and viral RNA template (37).
Applicant has now 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, in contrast to results obtained with deletion of the 54-nt segment, i.e.
pHIV/del-LD. The latter sequence was also independently involved in efficient expression of viral mRNA. 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.

Summary of the Invention This invention relates to an attenuated virus for use as a vaccine for the protection against HIV and in the prevention of acquired immunodeficiency syndrome (AIDS). The concept of an attenuated virus vaccine is that the virus would replicate to a limited extent or not at all, yet successfully immunize, and thereby protect, against subsequenst exposure to a live, virulent form of HIV.
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.

A 54-nt segment in the non-coding region of the HIV-l genome, downstream of the PBS, is important for viral replication in each of two principal ways. First, this region clearly 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, 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. This was shown through (a) studies in which transfection of cells using deleted DNA constructs failed to generate significant levels of viral mRNA, and (b) independent experiments 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.

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 PsS of HIV-l 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/WT (~) 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 (~) 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 CA 02208946 l997-06-26 of p24 content and used to infect MT-4 cells. Total cellular DNA (approximately 50 ~g) 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/WT, 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 2~M 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 ~-globin DNA as an internal control.
Fig. 5: Detection of minus-strand strong-stop DNA.
Infection by pHIV/del-LD1, 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
(20~g) from cells transfected with pHIV/del-LD; lane 2: RNA
(20~g) from cells transfected with pHIV/WT; lane 3: RNA (10 ~g) from cells transfected with pHIV/del-LD; lane 4: RNA
(lO~g) from cells transfected with pHIV/WT; lane 5: RNA
(20~g) 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 analyzed 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 infectlons 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 Re~lication 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 (PBS/del-7), and an extensive 54-nt deletion downstream of the PBS containing the aforementioned 7-nt segment (PBS/del-LD) (Fig. 1). In addition, the 54-nt deletion region was subdivided by smaller deletions termed pHIV/del-LD1, 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. 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 center 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 cellsa Viral ConstructInoculum p24 concn (nq/ml) on days (ng of p24) 7 10 13 30 90 pHIV/WT 50 23.5 27.3 I9.7b 18.9 16.8 13.9b pHIV/del-7 50 15.6 19.4 16.5b 12.7 21.9 17.9b pHIV/del-LD 50 1.5 2.0 2.4 2.0 1.8 0.4 1.1 1.3 1.2 1.3 pHIV/del-LD1 50 18.7 26.2 17.4b 18.5 20.8 16.8b pHIV/del-LD2 50 10.3 14.5 15.7b 8.6 11.4 13.2b pHIV/del-LD3 50 0.9 1.2 2.1 1.2 1.5 0.6 1.6 1.9 1.6 1.9 a MT-4 cells were infected with various viral constructs, and p24 levels 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 4-8 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 were unable to detect a DNA
signal (Fig. 3A, lanes 1, 2, 3 for wild-type, pHIV/del-7 and pHIV/del-LD, rspectively. 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 ~M 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 ~M AZT significantly impaired synthesis of DNA products generated after both the first and template switch events for each of the viruses tested (Figs. 4B,C) (compare lane 4 with lane 5, lanes 6 and 7, 8 and 9, 10 and 11, 12 and 13, 14 and 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, 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 (not shown). 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-LD1 (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 qene 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 CA 02208946 l997-06-26 patterns were observed in COS-7 cells transfected with pHIV/del-7 and wild type virus (not shown).
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, CA 02208946 l997-06-26 pHIV/del-LD1, pHIV/del-LD2, or pHIV/del-LD3 constructs (not shown).

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 tran~fected with pHIV/del-LD or pHIV/WT

~0 p24 level (ng/ml)a h after Intracellular Extracelllular transfection pHIV/del-LD pHIV/WT pHIV/del-LD pHIV/WT
tran~fection transfection transfection tran~fection 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 i 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 variou~ times after transfection, both intracellular and extracellular viral p24 levels were determined. Data are meanR + st~n~rd deviations from four separate experiments.

CA 02208946 l997-06-26 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-LD1, pHIV/del-LD2 or pHIV/del-LD3 constructs (not shown). 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.

Summary of the experiments Reverse transcription is initiated at the PBS to which the tRNA primer is bound. The HIV-1 PBS is located about 180-nt from the 5' terminus of unspliced RNA (23). The PBS is flanked by the R/U5 at its 5' border and by a 133-nt untranslated sequence at its 3' end (23). Increasing evidence suggests that the untranslated sequences that flank the PBS
are involved in several steps of viral replication, including reverse transcription, integration, expression of proviral genome, and packaging.
Two lines of data prompted applicant to initiate an identification of regions downstream of the PBS that are essential for viral replication. First, efficient retroviral reverse transcription requires interaction between primer tRNA
and the RNA at multiple sites (1,12,13,24,25,26,51). Second, HIV-1 has evolved to choose tRNALYs3 as a primer for optimal growth (16,36,58).
As an additional control, applicant also employed AZT, as described in the Examples, to block synthesis of viral DNA
products. Consistent with previous observations, applicant found that AZT had no effect on the presence of minus-strand strong-stop DNA, which is carried into cells by the virions in which it is made (39,54). However, the use of AZT efficiently interfered with production of DNA products that are generated after each of the first and second template switch events, consistent with previous observations (3).

Non-essentiality of a small, restricted region downstream of the PBS
A short 6-nt sequence, located immediately downstream of the PBS, was previously shown to be important in specifying CA 02208946 l997-06-26 utilization of the tRNA primer (33). Applicant has now shown that this sequence is apparently unnecessary for either synthesis of minus-strand strong-stop DNA in infected cells (pHIV/del-7) after transfection (Fig. 2) or for viral replication, consistent with in vi tro studies that employed nucleocapsid protein (37). Others have reported that nt substitutions downstream of the PBS may be observed in HIV-1 revertants that had been deleted within the PBS; these revertants appeared to have wild-type replication capacity (50). Thus, this small 6-7 nt sequence may not be required in either maintenance of viral secondary structure or interaction with tRNALYs3. Likewise, an 18 -nt segment, encompassing the above-mentioned 7-nt downstream of the PBS, is not essential for synthesis of viral DNA and viral replication, in spite of being DNase sensitive (17).
It should be noted that applicant's experiments were performed with a transient transfection system, while others employed "integrated templates" (17). While studies on integrated templates would have provided additional information, the results of Fig 2 show that virus infectivity was not severely impacted by deletion of a 7-nt stretch downstream of the PBS.

Identification of sequences im~ortant for viral replication downstream of the PBS
An extended 54-nt deletion, that does not compromise packaging/dimerization signals or the splice donor (2,11,15,35,41,52), was found to significantly diminish virus replication (pHIV/del-LD) (Fig.2). The proximity of this 54-nt region to the PBS might play a role in limiting viral replication, due to a defect in reverse transcription (37).
Measurement of reverse transcribed DNA, shortly after viral entry, revealed only low levels of product (Fig. 4). This was most pronounced when a 18-nt segment at the 3' end of the 54-nt region was removed (Fig. 5). This deficit in reverse transcription could be due to poor NCp-mediated interactions between tRNA primer and the RNA template (37).
In the case of minus-strand strong-stop DNA, it can be argued that the above experiments do not distinguish between synthesis of material that is present in virions versus that produced in cells after infection (54,39). For this reason, applicant employed AZT to influence the synthesis of viral DNA
products that are generated after the first template switch.
As previously demonstrated, applicant found that the use of AZT in its protocols did not result in chain termination until after the first template switch event (3,60). The potential role of contaminating DNA plasmids was also ruled out through mock infection protocols performed with DNA from cells inoculated with heat-inactivated viruses (see the Examples).
Applicant also found that deletion of the 54-nt region resulted in a significant decrease in accumulation of viral RNA transcripts in transfected cells (Fig. 6). The relationship between defects in reverse transcription and mRNA
production is unknown. The 54-nt segment could conceivably have affected both of these activities. Of course, the observed differences could be explained if pHIV/del-LD m-RNA

was less stable than wild-type viral RNA. However, applicant did not observe differences in stability between the various wild-type and mutated RNA transcripts studied. It is also unlikely that this region would influence efficiency of integration, since the latter process is primarily affected by the termini of reverse transcribed viral DNA (46,56). Indeed, MT-4 cells infected with the pHIV-1/del-LD virus contained integrated proviral DNA.
Applicant's results show that the 54-nt stretch downstream of the PBS is important for virus replication and that a 16-nt sequence, located at the 3' end of this segment, is principally involved. Further studies on nucleosome structure and/or identification of potential transcription factor-binding sites, through use of reporter genes, will yield additional functional information, as will further deletion analysis of the 16-nt segment itself (17,18,53,55).
Most binding elements for inducible or constitutive cellular transcripts are located upstream of the PBS (for review, see reference 13). However, untranslated sequences downstream of the PBS can also affect viral transcription activity as documented here.
It is also interesting that the 54-nt deletion had little effect on packaging of viral RNA, in spite of overlap at its 3' end with a substitution mutation that extends further downstream, and that can affect RNA encapsidation (32).
Differences between these constructs could have resulted in RNA structures that may have been differentially recognized by viral proteins involved in selection and encapsidation. The use of different viral strains and cell lines might also have contributed to differences between the studies.

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'-TGGCGCCCG AACAGGGACCGCGCACGGCAAGAGGCG-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 CsC12 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 ~m-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 MgC12 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 ~M 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 ~g 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 94OC (1 min), 60OC (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 ~g) 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 pBH10 viral DNA as a radiolabelled probe (Nick translation system, Life Technologies, Toronto, Canada) as described (10).
To quantitify 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 W irradiation (Amersham).
Hybridization reactions were performed as described for Northern blots (lo). 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 ~g/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 ~g RNA were used in reverse transcription reactions, using 5'-TTTATTGAGGCTTAAGCAGTGGG-3' (nt56 to nt78) as an antisense primer in a total volume of 20 ~l. One ~l 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 analyzed 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

1. A proviral DNA clone of human immunodeficiency virus (HIV) selectively deleted in regard to a nucleotide segment found immediately downstream of the primer binding sequence.

2. An attenuated virus generated using the proviral DNA
clone of claim 1.

3. A method of immunizing against acquired immunodeficiency syndrome comprising administering the attenuated virus of claim 2 to a human.