The present invention provides recombinant DNA viral vectors which co-express lentivirus genes encoding structural and enzymatic polypeptides capable of assembling into defective nonself-propagating viral particles. The viral DNA vectors as well as the viral particles can be used as immunogens and for targeted delivery of heterologous gene products and genes.

BACKGROUND 
Vaccination has played a key role in the control of viral diseases during 
the past 30 years. Vaccination is based on a simple principle of immunity: 
once exposed to an infectious agent, an animal mounts an immune defense 
that protects against infection by the same agent. The goal of vaccination 
is to induce the animal to mount the defense prior to infection. 
Conventionally, this has been accomplished through the use of live 
attenuated or killed forms of the virus as immunogens. The success of 
these approaches in the past has been due in part to the presentation of 
native antigen and the ability of attenuated virus to elicit the complete 
range of immune responses obtained in natural infection. However, 
conventional vaccine methodologies have always been subject to a number of 
potential limitations. Attenuated strains can mutate to become more 
virulent or non-immunogenic; improperly inactivated vaccines may cause the 
disease that they are designed to prevent. 
Recombinant DNA technology offers the potential for eliminating some of the 
limitations of conventional vaccines, by making possible the development 
of vaccines based on the use of defined antigens, rather than the intact 
infectious agent, as immunogens. These include peptide vaccines, 
consisting of chemically synthesized, immunoreactive epitopes; subunit 
vaccines, produced by expression of viral proteins in recombinant 
heterologous cells; and the use of live viral vectors for the presentation 
of one or a number of defined antigens. 
Both peptide and subunit vaccines are subject to a number of potential 
limitations. A major problem is the difficulty of ensuring that the 
conformation of the engineered proteins mimics that of the antigens in 
their natural environment. Suitable adjuvants and, in the case of 
peptides, carrier proteins, must be used to boost the immune response. In 
addition, these vaccines elicit primarily humoral responses, and thus may 
fail to evoke effective immunity. 
Some of the problems associated with the use of peptides and subunit 
vaccines can be overcome through the use of live viral vectors to present 
heterologous antigens. A number of viral vectors, including retro-, 
adeno-, herpes-, and poxviruses (Cepko et al., 1984. Cell 37:1053-1062; 
Morin et al., 1987. Proc. Natl. Acad. Sci. USA 84:4626-4630; Lowe et al., 
1987. Proc. Natl. Acad. Sci. USA 84:3896-3900; Panicali & Paoletti, 1982. 
Proc. Natl. Acad. Sci. USA 79:4927-4931 Mackett et al., 1982. Proc. Natl. 
Acad. Sci. USA 79:7415-7419) have been developed; the greatest effort has 
been concentrated on the development of vaccinia virus, an orthopox virus, 
as an infectious eukaryotic cloning vector for this purpose (Paoletti and 
Panicali, U.S. Pat. No. 4,603,112). Heterologous genes, including those 
encoding antigens from a variety of pathogens, have been expressed in the 
vaccinia vector system. In all cases, the foreign gene product expressed 
by the recombinant vaccinia virus was similar or identical to the gene 
product synthesized under native conditions. In some instances, 
vaccination of laboratory animals with recombinant vaccinia viruses has 
protected these animals against challenge with the correlate pathogens 
(Paoletti et al., 1984. Proc. Natl. Acad. Sci. USA 81:193-197; Kieny et 
al. 1984. Nature 312:163-166; Alizon et al., 1984. Nature 312: 757-760; 
Boyle et al. 1985. Gene 35: 169-177; Yilma et al. 1988. Science 
242:1058-1061). 
Recombinant approaches have been used in attempts to develop vaccines 
against diseases for which no vaccine currently exists, or for which 
conventional vaccine approaches are less desirable. For example, since the 
human immunodeficiency virus (HIV) was first identified as the etiologic 
agent of Acquired Immunodeficiency Disease Syndrome (AIDS), 
(Barre-Sinoussi et al., 1983. Science 220:868; Levey et al., 1984. Science 
225:840; Gallo et al., 1984. Science 224:500), considerable effort has 
been directed towards the development of a safe and effective vaccine. 
These efforts have relied upon a broad spectrum of strategies, ranging 
from the use of small synthetic peptides to whole inactivated virus as 
immunogen. 
The emergence of the AIDS pandemic may represent the most serious public 
health threat of the twentieth century. Since the recognition of AIDS in 
1981, extensive research has resulted in substantial advances in the 
understanding of the disease. The causative virus, (HIV), has been 
identified and the major routes of transmission have been shown to be 
sexual contact and exchange of blood products (Curran et al. 1985. Science 
229:1352). The nucleotide sequences of the genomes of many isolates of HIV 
have been determined and the molecular biology of the virus is under 
intensive investigation. However, much work remains to be done in 
elucidating viral replication in infected individuals and its role in the 
pathogenesis of disease. 
The human immunodeficiency viruses, HIV-1 and HIV-2, are members of the 
lentivirus subclass of retroviruses (Gonda et al., 1985. Science 227:173; 
Sonigo et al., 1985. Cell 42:369). Also in this subclass are the related 
simian immunodeficiency viruses (SIV; Daniel et al., 1985. Science 228: 
1201). The human and simian immunodeficiency viruses share similar 
morphology. The virus particles contain an inner core comprised of capsid 
proteins (encoded by the viral gag gene) that encase the viral RNA genome 
(Rabson & Martin, 1985. Cell 40:477). The central core is surrounded by a 
lipid envelope that contains the virally-encoded envelope glycoproteins. 
Virus-encoded enzymes required for efficient replication, such as the 
reverse transcriptase and integrase (encoded by the pol gene), are also 
incorporated into the virus particle. 
Nucleotide sequence analysis of HIV and SIV genomes has shown that these 
viruses also share a distinctive genome organization (FIG. 1), as well as 
considerable DNA sequence homology (Chakrabarti, et al., 1987. Nature 
328:543; Franchini et al., 1987. Nature 328:539). The approximately 10 kb 
genome comprises the flanking long terminal repeat (LTR) sequences that 
contain regulatory segments for viral replication, as well as the gag, 
pol, and env genes coding for the core proteins, the reverse 
transcriptase-protease-endonuclease, and the envelope glycoproteins, 
respectively. These viruses also contain at least six additional genes, 
some of which have known regulatory functions. 
Finally, the human and simian immunodeficiency viruses share common 
biological properties, including cytopathic effect, tropism for 
CD4-bearing cells, and, most importantly, the ability to induce long term 
persistent infection and chronic immunodeficiency disease in humans (HIV-1 
and HIV-2) or non-human primates (SIV). The similarities between HIV and 
SIV have led to the development of SIV infection of rhesus macaque monkeys 
as a model system for the study of pathogenesis and prevention of 
immunodeficiency disease. 
There are obvious difficulties with the use of whole virus for an HIV 
vaccine. The fear that an attenuated virus could revert to virulence, and 
the danger of incomplete inactivation of killed virus preparations, 
together with the reluctance to introduce the HIV genome into seronegative 
individuals have argued against the uses of live attenuated or killed HIV 
vaccines for the prevention of infection. 
AIDS vaccines now under consideration span the range of possible 
recombinant approaches; many, though not all, rely upon the use of all or 
part of the envelope glycoprotein as immunogen. However, many of the 
recombinant vaccines tested to date have yielded disappointing results. 
For example, when a subunit vaccine consisting of envelope glycoprotein 
gp120 was used to immunize chimpanzees, specific humoral immune responses 
to HIV capable of neutralizing the virus in vitro were elicited; however, 
the immunization failed to protect the animals from HIV infection (Berman 
et al., 1988. Proc. Natl. Acad. Sci. USA. 85:5200-5204). The use of live 
recombinant vaccinia virus expressing the HIV envelope glycoproteins as a 
vaccine for the prevention of HIV infection in chimpanzees has also been 
unsuccessful. Infection of susceptible cells with these recombinants 
results in the normal synthesis, glycosylation, processing, and membrane 
transport of the envelope polypeptide (Chakrabarti et al., 1986. Nature 
320:535; Hu et al., 1986. Nature 320:537). The gene product can be 
recognized by serum antibodies from patients with AIDS, and has been shown 
to mediate syncytia formation with cells expressing the CD4 cell surface 
epitope (Lifson et al., 1986. Nature 323:725). Vaccination of several 
species with these recombinants has elicited strain-specific humoral 
immune responses as well as cell-mediated responses (Hu, et al., 1986 
Nature 320:537; Chakrabarti, et al., 1986. Nature 320:535; Kieny et al., 
1986. Biotechnology 4:790; Zarling et al., 1986. Nature 323:344; Hu et 
al., 1987. Nature 328: 721; Zagury et al., 1987. Nature 326:249; Zagury et 
al., 1988. Nature 322:728). Nevertheless, despite these immune responses, 
these vaccines failed to protect chimpanzees from infection by HIV (Hu et 
al., 1987. Nature 328:721). The failure of these initial vaccinia-based 
vaccines may be attributed to a variety of factors. The immunogenicity of 
the vaccinia recombinants may not have been optimal; in fact, the 
recombinants elicited low antibody titers in chimpanzees. Certainly, the 
fact that these vaccines induce expression of only a single HIV-1 
polypeptide suggests that they may not have the maximum potential for 
stimulating protective immune responses. Furthermore, the use of 
chimpanzees to evaluate AIDS vaccines is itself questionable, as 
chimpanzees, although they can be infected with HIV, do not develop AIDS 
(Alter et al., 1984. Science 226:549; Fultz et al., 1986. J. Virol. 
58:116). 
The potential drawback to any of the recombinant approaches to AIDS vaccine 
development is that they have relied upon the use of a single HIV antigen, 
most usually the envelope glycoprotein, as immunogen. The success in the 
past of traditional vaccine approaches for other diseases (such as polio, 
measles, mumps, and rabies), which are based on the use of whole virus, 
either live attenuated or killed, as immunogens, suggests that antigen 
presentation is of paramount importance in eliciting protective immune 
responses. 
Advances in recombinant DNA technology may make it possible to use 
heterologous expression systems for the synthesis not only of individual 
antigens, but also of defective, non-self propagating, virus-like 
particles. It has been demonstrated that capsid proteins of certain 
viruses can assemble into particles morphologically and immunologically 
similar to the corresponding virus. For example, the P1 precursor of 
several picornaviruses synthesized in vitro can be processed into 
individual capsid proteins which then assemble into immunoreactive 
virion-like particles (Nicklin et al. 1986. Biotechnology 4:33; Palmenberg 
et al., 1979. J. Virol. 32:770; Shih et al., 1978. Proc. Natl. Acad. Sci. 
USA 75:5807; Hanecak et al., 1982. Proc. Natl. Acad. Sci. USA 79:3973; 
Grubman et al., 1985. J. Virol. 56:120). Self-assembly of capsid proteins 
expressed in vivo in several recombinant expression systems has also been 
reported. For example, when human hepatitis B surface antigen is expressed 
in yeast cells, the polypeptide assembles into particles similar in 
appearance to those isolated from human plasma (Valenzuela et al., 1982. 
Nature 298: 347); these particles stimulate anti-hepatitis B antibody 
production in several species and can protect chimpanzees from virus 
challenge (McAleer et al., 1984. Nature 307:178). In another example, it 
was shown that coexpression of canine parvovirus (CPV) capsid proteins VP1 
and VP2 in murine cells transformed with a bovine papilloma virus/CPV 
recombinant plasmid resulted in the formation of self-assembling 
virus-like particles (Mazzara et al., 1986. in Modern Approaches to 
Vaccines, Cold Spring Harbor Laboratory, N.Y.; R. M. Chanock and R. A. 
Lerner, eds. pp. 419-424; Mazzara et al., U.S. patent application Ser. No. 
905,299, filed Sep. 8, 1986); when used to vaccinate susceptible dogs, 
these empty capsids elicited immune responses capable of protecting 
against CPV challenge. Finally, it has been shown that the HIV-1 p55gag 
precursor polypeptide expressed in Spodoptera frugiperda cells using a 
baculovirus expression vector assembles into virus-like particles which 
are secreted into the cell culture medium (Gheysen et. al., 1988. Modern 
Approaches to New Vaccines, Cold Spring Harbor Laboratory, N.Y. September 
14-18, abstract no. 72). 
SUMMARY OF THE INVENTION 
This invention pertains to recombinant viral vectors capable of expressing 
at least two different polypeptides of a heterologous virus capable of 
self-assembly, in vivo or in vitro, into defective, non-self propagating 
viral particles, and to methods of producing the recombinant virus. This 
invention also pertains to intermediate DNA vectors which recombine with a 
parent virus in vivo or in vitro to produce the recombinant viral vector, 
and to methods of vaccinating a host with the recombinant viral vector to 
elicit protective immunity against the correlate heterologous pathogenic 
virus. In addition, this invention pertains to defective, non-self 
propagating viral particles, such as HIV, SIV, or picornaviral particles, 
produced by the recombinant viral vectors; these viral particles may be 
isolated and used themselves as immunogens for vaccination against 
pathogenic viruses, or for therapeutic purposes, such as enhancing immune 
responses in an infected individual, or for targeted delivery of 
therapeutic agents, such as cytotoxic drugs, to specific cell types.

DETAILED DESCRIPTION OF THE INVENTION 
1. Genes Encoding Viral Antigens 
Genes encoding viral polypeptides capable of self assembly into defective, 
non-self propagating viral particles can be obtained from the genomic DNA 
of a DNA virus or the genomic cDNA of an RNA virus or from available 
subgenomic clones containing the genes. These genes will include those 
encoding viral capsid proteins (i.e., proteins that comprise the viral 
protein shell) and, in the case of enveloped viruses, such as 
retroviruses, the genes encoding viral envelope glycoproteins. Additional 
viral genes may also be required for capsid protein maturation and 
particle self-assembly. These may encode viral proteases responsible for 
processing of capsid protein or envelope glycoproteins. 
As an example, the genomic structure of picornaviruses has been well 
characterized, and the patterns of protein synthesis leading to virion 
assembly are clear (Rueckert, R. in Virology (1985), B. N. Fields et al., 
eds. Raven Press, New York, pp 705-738). In picornaviruses, the viral 
capsid proteins are encoded by an RNA genome containing a single long 
reading frame, and are synthesized as part of a polyprotein which is 
processed to yield the mature capsid proteins by a combination of cellular 
and viral proteases. Thus, the picornavirus genes required for capsid 
self-assembly include both the capsid structural genes and the viral 
proteases required for their maturation. 
Another example of viruses from which genes encoding self-assembling capsid 
proteins can be isolated are the retroviruses HIV and SIV. The genomic 
structures of these viruses have been well characterized, and are 
represented diagrammatically in FIG. 1. The gene organization of the two 
viruses is remarkably similar, and homologs of each of the HIV structural 
genes are present in SIV. 
The HIV and SIV gag genes encode the capsid proteins. Like the picornaviral 
capsid proteins, the HIV and SIV gag proteins are synthesized as a 
precursor polypeptide that is subsequently processed, by a viral protease, 
into the mature capsid polypeptides. However, the gag precursor 
polypeptide can self-assemble into virus-like particles in the absence of 
protein processing (Gheysen et al., 1988. Modern Approaches to New 
Vaccines, Cold Spring Harbor Laboratory, N.Y. September 14-18, abstract 
no. 72). Unlike picornavirus capsids, HIV and SIV capsids are surrounded 
by a loose membranous envelope that contains the viral glycoproteins. 
These are encoded by the viral env gene. 
The examples illustrate the use of SIV and HIV genes selected for 
expression in recombinant viruses of this invention. These genes and their 
protein products are outlined in Table 1. The three major virion 
components derived from the env, gag, and pol genes are synthesized as 
precursor polyproteins which are subsequently cleaved to yield mature 
polypeptides as outlined in Table 1. 
TABLE 1 
______________________________________ 
SIV and HIV Genes for Recombination into Pox Virus 
Gene Gene Product.sup.a 
Processed Peptides 
______________________________________ 
env gp160 gp120 extracellular 
membrane protein 
gp41 transmembrane 
protein 
gag p55 p24 
p17 capsid proteins 
p15 
pol p160.sup.b p10 protease 
p66/p51 reverse 
transcriptase 
p31 endonuclease 
______________________________________ 
.sup.a Sizes given are for HIV proteins; size of the SIV polypeptides may 
be slightly different. 
.sup.b Part of the gagpol product. 
2. Parent Viruses 
A number of viruses, including retroviruses, adenoviruses, herpesviruses, 
and pox viruses, have been developed as live viral vectors for the 
expression of heterologous antigens (Cepko et al., 1984. cell 
37:1053-1062; Morin et al., 1987. Proc. Natl. Acad. Sci. USA 84:4626-4630; 
Lowe et al., 1987. Proc. Natl. Acad. Sci. USA 84:3896-3900; Panicali & 
Paoletti, 1982. Proc. Natl. Acad. Sci. USA 79: 4927-4931; Mackett et al., 
1982. Proc. Natl. Acad. Sci. USA 79:7415-7419). The examples given 
illustrate the use of the pox virus family. The preferred pox virus is 
vaccinia virus, a relatively benign virus which has been used for years as 
a vaccine against smallpox. Vaccinia virus has been developed as an 
infectious eukaryotic cloning vector (Paoletti and Panicali, U.S. Pat. No. 
4,603,112) and recombinant vaccinia virus has been used successfully as a 
vaccine in several experimental systems. The virus is considered 
nononcogenic, has a well-characterized genome, and can carry large amounts 
of foreign DNA without loss of infectivity (Mackett, M. and G. L. Smith, 
1986. J. Gen. Virol. 67:2067). 
3. DNA Vectors for in Vivo Recombination With a Parent Virus 
According to the method of this invention, viral genes that code for 
polypeptides capable of assembly into viral particles are inserted into 
the genome of a parent virus in such as manner as to allow them to be 
expressed by that virus along with the expression of the normal complement 
of parent virus proteins. This can be accomplished by first constructing a 
DNA donor vector for in vivo recombination with a parent virus. 
In general, the DNA donor vector contains the following elements: 
i) a prokaryotic origin of replication, so that the vector may be amplified 
in a prokaryotic host; 
ii) a gene encoding a marker which allows selection of prokaryotic host 
cells that contain the vector (e.g., a gene encoding antibiotic 
resistance); 
iii) at least two heterologous viral genes (e.g., SIV, HIV, or picornavirus 
genes), each gene located adjacent to a transcriptional promoter capable 
of directing the expression of the gene; and 
iv) DNA sequences homologous to the region of the parent virus genome where 
the foreign gene(s) will be inserted, flanking the construct of element 
iii. 
Methods for constructing donor plasmids for the introduction of multiple 
foreign genes into pox virus are described in U.S. patent application Ser. 
No. 910,501, filed Sep. 23, 1986, entitled "Pseudorabies Vaccine", which 
corresponds to EPO 0261940 the techniques of which are incorporated herein 
by reference. In general, all viral DNA fragments for construction of the 
donor vector, including fragments containing transcriptional promoters and 
fragments containing sequences homologous to the region of the parent 
virus genome into which foreign genes are to be inserted can be obtained 
from genomic DNA or cloned DNA fragments. The donor plasmids can be mono-, 
di-, or multivalent (i.e., can contain one or more inserted foreign gene 
sequences). 
The donor vector preferably contains an additional gene which encodes a 
marker which will allow identification of recombinant viruses containing 
inserted foreign DNA. Several types of marker genes can be used to permit 
the identification and isolation of recombinant viruses. These include 
genes that encode antibiotic or chemical resistance (e.g., see Spyropoulos 
et al., 1988, J. Virol. 62:1046; Falkner and Moss., 1988, J. Virol. 
62:1849; Franke et al., 1985. Mol. Cell. Biol. 5:1918), as well as genes, 
such as the E. coli lacZ gene, that permit identification of recombinant 
viral plaques by calorimetric assay (Panicali et al., 1986. Gene 
47:193-199). 
A method for the selection of recombinant vaccinia viruses relies upon a 
single vaccinia-encoded function, namely the 29K host-range gene product 
(Gillard et al. 1986. Proc. Natl. Acad. Sci. USA. 83:5573). This method 
was described in U.S. patent application Ser. No. 205,189, filed Jun. 20, 
1988, entitled "Methods of Selecting for Recombinant Pox Viruses", which 
corresponds to WO 89/12103 the teachings of which are incorporated herein 
by reference. Briefly, a vaccinia virus that contains a mutation in the 
29K gene, which is located in the HindIII K/M regions of the viral genome, 
is used as the host virus for in vivo recombination. The 29K mutation 
prevents the growth of this virus on certain host cells, for example, RK13 
(rabbit kidney) cells. The donor plasmid for insertion of foreign genes 
contains vaccinia DNA sequences capable of restoring the mutant gene 
function; these sequences also direct recombination to the site of the 
mutant gene in the HindIII M region. Thus, recombinant vaccinia viruses 
regain the ability to grow in RK13 cells, and can be isolated on this 
basis from the non-recombinant parental viruses, which are unable to grow 
on these cells. 
A preferred DNA vector for recombination with the preferred vaccinia virus 
comprises: 
a. one or more transcriptional promoters (e.g., the vaccinia 7.5K, 30K, 
40K, 11K or BamF promoters or modified versions of these promoters), 
capable of directing expression of adjacent genes in vaccinia virus, each 
linked to; 
b. one or more structural genes encoding viral antigens of interest, each 
under the control of a transcriptional promoter; 
c. a marker for the selection of recombinant parent virus, which may 
comprise: 
(1) a transcriptional promoter (e.g., the BamF promoter of vaccinia virus) 
linked to a gene encoding a selectable marker (e.g., the E. coli lacZ 
gene); or 
(2) parent virus structural gene sequences which restore a viral host-range 
or other virus growth promoting function (e.g., the 29K polypeptide of 
vaccinia); 
d. DNA sequences homologous with a region of the parent virus flanking the 
construct of elements a-c (e.g., the vaccinia TK or HindIII M sequences); 
e. a vector backbone for replication in a prokaryotic host including a 
marker for selection of bacterial cells transformed with the plasmid 
(e.g., a gene encoding antibiotic resistance). 
4. Integration of Foreign DNA Sequences into the Viral Genome and Isolation 
of Recombinants 
Homologous recombination between donor plasmid DNA and viral DNA in an 
infected cell results in the formation of recombinant viruses that 
incorporate the desired elements. Appropriate host cells for in vivo 
recombination are generally eukaryotic cells that can be infected by the 
virus and transfected by the plasmid vector. Examples of such cells 
suitable for use with a pox virus are chick embryo fibroblasts, HuTK143 
(human) cells, and CV-1 and BSC-40 (both monkey kidney) cells. Infection 
of cells with pox virus and transfection of these cells with plasmid 
vectors is accomplished by techniques standard in the art (Panicali and 
Paoletti, U.S. Pat. No. 4,603,112). 
Following in vivo recombination, recombinant viral progeny can be 
identified by one of several techniques. For example, if the DNA donor 
vector is designed to insert foreign genes into the parent virus thymidine 
kinase (TK) gene, viruses containing integrated DNA will be TK.sup.- and 
can be selected on this basis (Mackett et al., 1982, Proc. Natl. Acad. 
Sci. USA 79:7415). Alternatively, co-integration of a gene encoding a 
marker or indicator gene with the foreign gene(s) of interest, as 
described above, can be used to identify recombinant progeny. One 
preferred indicator gene is the E. coli lacZ gene: recombinant viruses 
expressing beta-galactosidase can be selected using a chromogenic 
substrate for the enzyme (Panicali et al., 1986, Gene 47:193). A second 
preferred indicator gene for use with recombinant vaccinia virus is the 
vaccinia 29K gene: recombinant viruses that express the wild type 29K 
gene-encoded function can be selected by growth on RK13 cells. 
As described more fully in the Examples, monovalent and divalent donor 
plasmids containing HIV or SIV genes were recombined into vaccinia at 
either the TK gene or the HindIII M region and recombinant viruses were 
selected as described above. 
5. Characterizing the Viral Antigens Expressed by Recombinant Viruses 
Once a recombinant virus has been identified, a variety of methods can be 
used to assay the expression of the polypeptide encoded by the inserted 
gene. These methods include black plaque assay (an in situ enzyme 
immunoassay performed on viral plaques), Western blot analysis, 
radioimmunoprecipitation (RIPA), and enzyme immunoassay (EIA). Antibodies 
to antigens expressed by viral pathogens are either readily available, or 
may be made according to methods known in the art. For example, for human 
or simian immunodeficiency viruses, the antibodies can be (a) for parent 
virus/SIV recombinants, sera from macaque monkeys infected with SIV; and 
(b) for parent virus/HIV recombinants, either sera from human patients 
infected with HIV, or commercially available monoclonal antibodies 
directed against specific HIV polypeptides. 
6. Viral Particle Formation 
Expression analysis described in the preceding section can be used to 
confirm the synthesis of the polypeptides encoded by inserted heterologous 
viral genes, but does not address the question of whether these 
polypeptides self-assemble, in vivo or in vitro, into defective viral 
particles. Two experimental approaches can be used to examine this issue. 
The first approach is to visually examine by electron microscopy lysates of 
cells infected with recombinant viruses that express one or more viral 
polypeptides. In the experiments reported below, for example, vaccinia 
recombinants that express gag, env+gag, or env+gag+pol genes of SIV and/or 
HIV gave rise to the formation of defective retroviral particles; in cells 
infected with the recombinants that coexpress gag and env polypeptides, 
the particle envelope contained the envelope glycoproteins as shown by the 
presence of glycoprotein "spikes" on the surface of the particles. The 
presence of retroviral envelope glycoproteins on the surface of the 
particles can be demonstrated with immunogold electron microscopy, using a 
monoclonal antibody directed against one of the envelope glycoproteins. 
In order to further characterize the defective viral particles produced by 
recombinant viruses expressing viral polypeptides, these particles can be 
isolated by high speed centrifugation from the culture medium of cells 
infected with the recombinant viruses in the presence of .sup.35 
S!-methionine. The pellet resulting from centrifugation of the culture 
medium can be resuspended and both the pellet and the supernatant can be 
immunoprecipitated with an appropriate antiserum to analyze the viral 
polypeptides present in each fraction. For example, in the case of 
recombinants expressing HIV or SIV polypeptides, either human anti-HIV 
antisera (for vaccinia/HIV recombinants) or macaque anti-SIV antisera (for 
vaccinia/SIV recombinants) can be used for these analyses. 
In the case of recombinant viruses that coexpress HIV or SIV env and gag 
polypeptides, the pellet will contain both envelope and core proteins if 
retroviral particles are formed. As described in the examples, when this 
experiment was performed using vaccinia recombinants that coexpress HIV or 
SIV env and gag polypeptides, the pellet contained both env and gag 
polypeptides, as would be expected if these polypeptides were assembling 
into defective retroviral particles. By contrast, the supernatant 
contained only the envelope glycoprotein gp120. 
To further characterize the material in the pellet resulting from 
centrifugation of the culture medium, the pellet can be resuspended and 
analyzed on a sucrose gradient. The gradient can then be fractionated and 
the fractions immunoprecipitated with the appropriate antiserum. These 
experiments show whether the pellet contains material banding at the 
density expected for defective viral particles. 
These methods can also be used to determine whether expression of viral 
polypeptides directed by two different viruses present in the same 
infected cell gives rise to the production of defective viral particles. 
For example, these experiments can be performed using cells coinfected in 
vitro with one recombinant expressing gag and a second recombinant 
expressing env. The simultaneous expression in a single cell of both env 
and gag polypeptides, whether directed by a single divalent recombinant 
virus or by two different monovalent viruses, would be expected to result 
in the formation of defective retroviral particles that contain a protein 
core comprising gag polypeptides surrounded by an envelope containing 
virally-encoded envelope glycoproteins. 
7. Vaccines 
Live recombinant viral vectors that express heterologous viral antigens 
capable of self-assembly into defective non-self-propagating virus 
particles can be used to vaccinate humans or animals susceptible to 
infection if the viral vector used to express the heterologous defective 
virus particles infects but does not cause significant disease in the 
vaccinated host. Examples of such benign viral vectors include certain pox 
viruses, adenoviruses, and herpes viruses. For example, vaccination with 
live recombinant vaccinia virus is followed by replication of the virus 
within the host. During replication, the viral genes are expressed along 
with the normal complement of recombinant virus genes. Thus, during the 
two-week post-immunization period when the live recombinant virus is 
replicating (Fenner, F., in Virology, Fields et al., eds. Raven Press, New 
York, 1985, pp 661-684), viral antigens may be presented to the host 
immune system in a manner that closely mimics the presentation of antigens 
in an authentic viral infection, that is, as defective, 
non-self-propagating viral particles extremely similar to the native 
virus. Viral antigens repeatedly presented both as free particles and in 
association with recombinant virus-infected cells may have the potential 
to prime the immune system to recognize and eliminate the virus during the 
early events of viral infection. 
Alternatively, the defective virus particles produced by these recombinant 
vector viruses can be isolated from cells infected in vitro with the 
recombinant vector viruses and from the culture medium of these infected 
cells, and themselves used for vaccination of individuals susceptible to 
viral infection. These particles resemble the native virus, but will not 
contain infectious viral genetic material. Consequently, they offer the 
advantage of conventional killed virus vaccine preparations: the ability 
authentically to present immunogenic antigens to the immune system of the 
vaccinated host. At the same time such particles circumvent the major 
drawbacks to the use of killed virus as a vaccine for the prevention of 
infection, including the danger of incomplete inactivation of killed virus 
preparations and, as for,the case of certain viruses, such as 
retroviruses, the reluctance to introduce a complete viral genome (the HIV 
genome, for example) into seronegative individuals. 
Vaccine compositions utilizing these defective virus particles would 
generally comprise an immunizing amount of the viral particles in a 
pharmaceutically acceptable vehicle. The vaccines would be administered in 
a manner compatible with the dosage formulation, and in such amount as 
would be therapeutically effective and immunogenic. 
Finally, the purified particles may be used in combination with live 
recombinant viruses as part of a total vaccination protocol, either as the 
primary immunizing agent, to be followed by vaccination with live 
recombinant virus, or to boost the total immune response after primary 
vaccination with live recombinant virus. 
8. Therapeutic Use of Recombinant Viruses Expressing Viral Antigens Capable 
of Assembling into Defective Viral Particles; Therapeutic Use of Defective 
Viral Particles Produced by These Recombinant Viruses 
Even if immunization cannot protect against infection, immunization of a 
previously infected individual might prolong the latency period of that 
virus within the individual. This may be particularly important in the 
case of viral infections characterized by long latency periods, such as 
HIV infection. The long incubation time between HIV infection and the 
development of clinical AIDS may be due to an immune response to the 
initial infection which persists with health and wanes with disease. If 
this is the case, boosting the immune response by immunization with HIV 
antigen/parent virus recombinants that produce retroviral-like particles, 
or alternatively, with the purified particles themselves, may prevent the 
development of disease and reduce contagiousness (Salk, 1987. Nature 
327:473). 
9. Therapeutic Use of Defective Virus Particles as Agents for Targetted 
Drug Delivery 
Defective, non-self-propagating virus particles can also be used to deliver 
certain drugs (e.g. cytotoxic drugs, antiviral agents) to virus 
receptor-bearing cells. Such drugs may be coupled, by techniques known in 
the art, to the outer surface of the virus particle, or incorporated 
within, and delivered with high specificity to target cells. For example, 
cytotoxic drugs may be coupled to defective HIV particles and delivered 
with a high degree of specificity to CD4.sup.+ T cells, since the HIV 
envelope glycoprotein present on these particles binds specifically and 
with high affinity to the CD4 molecule. Similarly, poliovirus particles, 
for example, preferentially bind cells of the nasopharynx and gut, and 
thus can be used to direct delivery of specific agents to these cells. 
10. Diagnostic Uses of Virus-Like Particles 
Immunogenic virus-like particles can be used to diagnose viral infection. 
The particles can be used to raise a panel of monoclonal antibodies and 
polyclonal antisera which recognize various epitopes on the virion. These 
monoclonal and/or polyclonal antibodies can be used individually or 
together as capture antibodies for an immunoassay to detect the presence 
of virus in urine, blood, or feces. 
Alternately, the particles themselves can be used as antigens for an 
immunoassay to detect the presence of antibody in urine, blood, or feces. 
Particularly preferred immunoassays are solid phase immunometric assays 
(enzymetric radiometric). In such assays, the virus-like particle is 
immobilized on a solid phase to provide an immunoadsorbent. The techniques 
for use of solid phase immunoadsorbents are known in the art. 
This invention is illustrated further by the following examples: 
EXAMPLES 
Materials and Methods 
Cells and Virus 
E. coli strain MC1061 (Casadaban and Cohen, 1980, J. Mol. Biol. 138:179) 
was used as the host for the growth of all plasmids. The monkey kidney 
cell line BSC-40 (Brockman & Nathans, 1974. Proc. Natl. Acad. Sci. USA 
71:942), the thymidine kinase-deficient (TK-) human cell line Hu143TK- 
(Bacchetti and Graham, 1977. Proc. Natl. Acad. Sci. USA 74: 1590) and the 
rabbit kidney cell line RK13 (ATCC #CCL37; Beale et al., 1963, Lancet 
2:640) were used for vaccinia infections and transfections. 
Vaccinia virus strain NYCBH (ATCC #VR-325) and 29K- lacZ+ strain vABT33 
(see U.S. patent application Ser. No. 205,189, filed Jun. 10, 1988, which 
corresponds to WO89/12108 the teachings of which are incorporated herein 
by reference) were used as the parental viruses for in vivo recombination. 
Enzymes 
Restriction enzymes were obtained from New England BioLabs or 
Boehringer-Mannheim. The large fragment of DNA polymerase (Klenow) was 
obtained from United States Biochemical Corp., T4 DNA polymerase was 
obtained from New England BioLabs, and T4 DNA ligase was obtained from 
Boehringer-Mannheim. 
Molecular Cloning Procedures 
Restriction enzyme digestions, purification of DNA fragments and plasmids, 
treatment of DNA with Klenow, T4 DNA polymerase, ligase, or linkers and 
transformation of E. coli were performed essentially as described 
(Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring 
Harbor Laboratory, Cold Spring Harbor, N.Y., 1982, incorporated herein by 
reference). 
Oligonucleotide mutagenesis was performed using synthetic oligonucleotides 
obtained from the Biology Department, Brandeis University, with reagents 
supplied by Amersham and used according to the manufacturer's 
instructions. 
Preparation of Vaccinia Virus Recombinants 
Viral infection, transfections, plague purification and virus amplification 
were performed essentially as described (Spyropoulos et al., 1988, J. 
Virol. 62:1046). 29K+ recombinants were selected and purified on RK13 
cells (see U.S. patent application Ser. No. 205,189, filed Jun. 10, 
1988which corresponds to WO89/12103, the teachings of which are 
incorporated by reference herein). TK.sup.- recombinants were selected 
and purified in the presence of 50 uM bromodeoxyuridine. 
Vaccinia Virus Genomic Analysis 
DNA was extracted from vaccinia virus-infected cells as described (Esposito 
et al., 1981, J. Virol. Methods 2:175) and analyzed by restriction enzyme 
digestions and Southern hybridization as described (Maniatis et al., 
Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, 
Cold Spring Harbor, N.Y., 1982). 
Protein Analysis 
Black plaque assay and immunoprecipitation analysis were performed 
essentially as described in (Smith et al., 1983. Proc. Natl. Acad. Sci. 
USA 80:7155 and Wittek et al., 1984. J. Virol 49:371); See also, U.S. 
patent application Ser. No. 910,501 filed Sep. 23, 1986 which corresponds 
to EP0261940 incorporated by reference herein. For black plaque and 
immunoprecipitation assays, macaque polyclonal antiserum against whole 
SIV, or human antiserum against whole HIV, were used. For 
immunoprecipitation analysis, vaccinia-infected cells were labelled either 
with .sup.3 H!-glucosamine or .sup.35 S!-methionine. 
Biochemical Analysis of Recombinant Vaccinia-directed Retroviral Particle 
Formation 
BSC-40 cells infected with the wild type or recombinant vaccinia virus were 
labeled with .sup.35 S!-methionine, using the same labeling procedure 
used for immunoprecipitation analysis. After 16-18 hours, the medium from 
infected cells was collected and clarified by centrifugation at 1000 rpm 
for 5 minutes. The resulting supernatant was centrifuged at 24K for 90 
minutes in an SW28 rotor. The supernatant was removed, and the resulting 
pellet was resuspended in 3 ml PBS buffer (136 mM NaCl, 2.7 mM KCl, 8.1 mM 
Na.sub.2 HP0.sub.4, 1.5 mM KH.sub.2 PO.sub.4). Samples from the 
supernatant and pellet were subjected to immunoprecipitation analysis, 
using macaque anti-SIV antiserum, or human anti-HIV antiserum as described 
in the preceding section. 
Analysis of Recombinant Vaccinia-directed Retroviral Particle Formation by 
Electron Microscopy 
BSC-40 cells were infected with wild type or recombinant vaccinia virus at 
a multiplicity of 10 for 16-18 hours as described above. Medium was then 
removed, and infected cells were washed twice in 0.2% gelatin in PBS 
buffer. 
For immunogold staining, samples were preincubated in 10% goat serum in 
PBS/gelatin for 30 minutes at 4.degree. C., then incubated in the 
appropriate monoclonal antibody diluted in PBS/gelatin for 60 minutes. 
Samples were then washed in PBS/gelatin twice and incubated with a 1:20 
dilution of goat anti-mouse IgG conjugated with gold (5nm) in PBS with 1% 
fetal calf serum, 0.1% sodium azide, and 5% human antibody serum for 60 
minutes. Cells were again washed in PBS/gelatin. 
Untreated or immunogold stained cells were fixed in 0.25% glutaraldehyde in 
0.1M sodium cacodylate buffer (pH 7.2) overnight, then washed in 0.1M 
sodium cacodylate buffer (pH 7.2) twice. Samples were then fixed in 1% 
OsO.sub.4 /0.1M cacodylate buffer (pH 7.2) for 11/2 hours and then washed 
twice in 0.1M cacodylate buffer (pH 7.2). Samples were then dehydrated in 
the following graded alcohols for 10 minutes each: 50, 70, 80, 95, 100 
(3X). Samples were then treated with propylene oxide twice for five 
minutes each, then overnight in uncapped vials in propylene oxide (4 
parts)/epox812 (6 parts). Samples were then embedded in epox812 and cured 
for 36 hours. 
Sections were cut on Sorval porter Blum MT-2 ultramicrotome at 1000 A, 
stained with alcoholic uranyl acetate and Sato's lead stain (Sato, T. 
1968. J. Elect. Mic. (Japan) 17:158-159), and viewed on a JEOL electron 
microscope. 
EXAMPLE 1 
Construction of Recombinant Plasmids Containing SIV Genes 
This example illustrates the construction of recombinant plasmids 
containing SIV genes for in vivo recombination with vaccinia virus (in 
vivo recombination (IVR) vectors). The construction and structure of 
plasmids pAbT4532B, pAbT4533, pAbT4536, pAbT4537, pAbT4574, pAbT4578, 
pAbT4582B, pAbT4583 and pAbT4589 is described in U.S. patent application 
Ser. No. 205,454, filed Jun. 10, 1988 which corresponds to WO 89/12095. 
The construction and structure of plasmid pAbT4027 is described in U.S. 
patent application Ser. No. 910,501, filed Sep. 23, 1986 which corresponds 
to EP 0261940. The construction and structure of plasmid pAbT4587 is 
described in U.S. patent application Ser. No. 229,343, filed Aug. 5, 1988 
which corresponds to WO90/01546. The teachings of the above- mentioned 
patent applications are incorporated by reference herein. 
pAbT4592 (FIG. 2): pAbT4578 (See U.S. patent application Ser. No.205,454, 
filed Jun. 10, 1988 which corresponds to WO 90/01546) was digested with 
KpnI and EcoRI, and a 2221 base pair (bp) fragment was isolated. This 
fragment was ligated to pAbT4587, which had been digested with KpnI and 
EcoRI, to give the plasmid pAbT4592. pAbT4592 is a vector for the 
insertion and expression of SIVmac251 gag and protease in vaccinia virus. 
pAbT4592 contains the gag-prot gene under the control of the vaccinia 40K 
promoter, flanked by vaccinia HindIII M region. The vector DNA includes 
the 29K host-range gene for selection of vaccinia recombinants and a 
bacterial replicon and ampicillin-resistance gene for growth and selection 
in E. coli. 
pAbT4593 (FIG. 3): pAbT4574 (U.S. patent application Ser. No.205,454) was 
digested with BamHI, and a 2589 bp fragment was isolated. This fragment 
was ligated to pAbT4587, which had been digested with BamHI and treated 
with calf alkaline phosphatase (CIP), to give the plasmid pAbT4593. 
pAbT4593 is a vector for the insertion and expression of SIVmac251 env in 
vaccinia virus. pAbT4593 contains the env gene under the control of the 
vaccinia 40K promoter, flanked by vaccinia DNA for directing recombination 
into the vaccinia HindIII M region. The vector DNA includes the 29K 
host-range gene for selection of vaccinia recombinants and a bacterial 
replicon and ampicillin-resistance gene for growth and selection in E. 
coli. 
pAbT4597 (FIG. 4): pAbT4578 (U.S. patent application Ser. No.205,454 which 
corresponds to WO89/12095) was digested with SacI, and the digested DNA 
was treated first with T4 DNA polymerase, and then with CIP. pAbT4589 was 
digested with SphI, then treated with T4 DNA polymerase, and finally 
digested with SmaI. A 2750 bp fragment was isolated from the digested 
pAbT4589; this fragment was ligated to the SacI digested pAbT4578 to yield 
pAbT4597. pAbT4597 is a vector for the insertion and expression of 
SIVmac251 env and gag-prot in vaccinia virus. pAbT4573 contains the 
gag-prot gene under the control of the vaccinia 30K promoter and the env 
gene under the control of the vaccinia 40K promoter. These genes are 
flanked by vaccinia DNA for directing recombination into the vaccinia 
HindIII M region. The vector DNA includes the 29K host-range gene for 
selection of vaccinia recombinants and a bacterial replicon and 
ampicillin-resistance gene for growth and selection in E. coli. 
EXAMPLE 2 
Construction of Recombinant Plasmids Containing HIV Genes 
pSVHenv and PBF128 are plasmids that contain portions of the HIV-1 strain 
B10 genome; these were obtained from E.I. Dupont de Nemours and Company. 
pHXBc2 is a plasmid that contains portions of the HIV-1 strain HSB2 
genome, it was obtained from Dr. Joseph Sodroski of the Harvard Medical 
School. The construction and structure of plasmids pAbT4532B and pAbT4554 
were described in U.S. patent application Ser. No. 205,454, filed Jun. 10, 
1988 which corresponds to WO89/12089. The construction and structure of 
plasmid pAbT4007 is described in U.S. patent application Ser. No. 910,501, 
filed Sep. 23, 1986 which corresponds to EP 0261940. The teachings of the 
above-mentioned patent applications are incorporated by reference herein. 
pAbT164 (FIG. 5) Plasmid pBF128 was digested with NcoI and BamHI and 
treated with the Klenow fragment of DNA polymerase; a 1700 bp fragment 
containing the HIV-1 gag gene was isolated from this digest. This fragment 
was ligated to plasmid pAbT4532B which had been digested with SmaI and 
treated with CIP, to give the plasmid pAbT164. 
pAbT164 is a vector for the insertion and expression of HIV-1 gag in 
vaccinia. pAbT164 contains the HIV gag gene under the control of the 
vaccinia 7.5K promoter, the DNA regions flanking the vaccinia TK gene for 
directing recombination in vaccinia, the lacZ gene under the control of 
the vaccinia BamF promoter for selection of vaccinia recombinants and a 
bacterial replicon and ampicillin-resistance gene for growth and selection 
in E. coli. 
pAbT4603 (FIG. 6) 
Plasmid pSVHenv was digested with XbaI and XhoI and treated with the Klenow 
fragment of DNA polymerase; a 2700 bp fragment containing the HIV-1 env 
gene was isolated from this digest. This fragment was ligated to plasmid 
pAbT4007 which had been digested with SmaI and treated with CIP, to give 
the plasmid pAbT167. 
pAbT167 was digested with KpnI and XbaI and a 225 bp fragment containing 
the 5' end of the HIV-1 env gene was isolated from this digest. This 
fragment was ligated to m13mp18 bacteriophage DNA (New England BioLabs) 
which had been digested with XbaI and KpnI and treated with CIP, to create 
pAbT220. The 5' end of the env gene was modified to remove most of the 5' 
non-coding sequences by oligonucleotide-directed mutagenesis, as described 
in Materials and Methods. Using the oligonucleotide 
5'-GAAAGAGCAGTAGACAGTGG-3' (Biology Department, Brandeis University), an 
AccI site was inserted approximately 10 bp upstream from the ATG 
initiation codon of the env coding sequence, creating pAbT221. pAbT221 was 
digested with EcoRI and HindIII, and an approximately 230 bp fragment was 
isolated. This was ligated to the plasmid pEMBL18+ (Dente et al., 1983. 
Nucl. Acids Res. 11:1645) which had been digested with EcoRI and HindIII 
and treated with CIP, to give the plasmid pAbT8536. 
pAbT8536 was digested with AccI, treated with the Klenow fragment of DNA 
polymerase, and then digested with KpnI. A 138 bp fragment containing the 
mutagenized 5' non-coding region of the env gene was isolated from this 
digest. The plasmid pAbT167 was digested with KpnI and SadI, and a 2461 bp 
fragment containing most of the env gene sequence was isolated. The 138 bp 
fragment from pAbT8536 and the 2461 bp fragment from pAbT167 were ligated 
to pAbT4554 which had been digested with SmaI and SacI and treated with 
CIP, to yield the plasmid pAbT8539. 
pAbT8539 was digested with EcoRI and a 2682 bp fragment containing the env 
gene was isolated. This was ligated to pAbT4587 which had been digested 
with EcoRI and treated with CIP, to yield the plasmid pAbT4603. 
pAbT4603 is a vector for the insertion and expression of HIV-1 env in 
vaccinia virus. pAbT4603 contains the env gene under the control of the 
vaccinia 40K promoter, flanked by vaccinia DNA for directing recombination 
into the vaccinia HindIII M region. The vector DNA includes the 29K 
host-range gene for selection of vaccinia recombinants and a bacterial 
replicon and ampicillin-resistance gene for growth and selection in E. 
coli. 
pAbT621 (FIG. 7) 
7A. Construction of plasmid pAbT598 (FIG. 7a). A plasmid derived from 
pHXBc2 (Sodroski et al., Nature 322:470 (1986)) was digested with BglI and 
SacI, releasing a fragment corresponding to nucleotide positions 715-6002. 
This DNA was then treated with T4 DNA polymerase. The 5287 bp fragment 
containing the HIV-1 gag and pol genes was isolated from this digest and 
ligated to pAbT4536 which had been digested with SmaI and treated with 
CIP, to yield the plasmid pAbT599. 
pAbT599 was digested with NdeI, treated with T4 DNA polymerase, then 
further digested with BglII, and a 3027 bp fragment was isolated from this 
digest. This fragment was ligated to an 8864 bp fragment isolated after 
digestion of pAbT164 with KpnI, treatment with T4 DNA polymerase, and 
further digestion with BglII, to yield plasmid pAbT598. 
B. Construction of pAbT621 (FIG. 7b). pAb4603 was digested with PstI, 
treated with T4 DNA polymerase, and further digested with Sal I, and a 
6326 bp fragment resulting from this digestion was isolated. pAbT598 was 
digested with BamHI and BalI, and a 1838 bp fragment was isolated. 
pAbT4554 was digested with SalI and BamHI, and a 420 bp fragment was 
isolated. These three fragments were ligated together to create pAbT621. 
pAbT621 is a vector for the insertion and expression of HIV-1 env and 
gag-prot in vaccinia. pAbT621 contains the HIV env gene under the control 
of the vaccinia 40K promoter, the SIV gag-prot gene under the control of 
the vaccinia 30K promoter. These genes are flanked by vaccinia DNA for 
directing recombination into the vaccinia HindIII M region. The vector DNA 
includes the 29K host-range gene for selection of vaccinia recombinants 
and a bacterial replicon and ampicillin-resistance gene for growth and 
selection in E.coli. 
EXAMPLE 3 
Construction of Recombinant Vaccinia Viruses Containing HIV-1 or SIVmac251 
Genes Under the Control of Vaccinia Promoters. 
In vivo recombination is a method whereby recombinant vaccinia viruses are 
created (Nankano et al., 1982. Proc. Natl. Acad. Sci. USA 79:1593; 
Paoletti and Panicali, U.S. Pat. No. 4,603,112). These recombinant viruses 
are formed by transfecting DNA containing a gene of interest into cells 
which have been infected by vaccinia virus. A small percent of the progeny 
virus will contain the gene of interest integrated into a specific site on 
the vaccinia genome. These recombinant viruses can express genes of 
foreign origin (Panicali and Paoletti, 1982, Proc. Natl. Acad. Sci. USA 
79:4927; Panicali et al., 1983. Proc. Nati. Acad. Sci. USA 80:5364). 
To insert SIVmac251 or HIV-1 genes into the vaccinia virus genome at the 
HindIII M region, a selection scheme based upon the 29K host-range gene, 
which is located in this region (Gillard et al., 1986. Proc. Natl. Acad. 
Sci. USA 83:5573) was used. Recombinant vaccinia virus vAbT33 contains the 
lacZ gene in place of a portion of the 29K gene. This lacZ insertion 
destroys the function of the 29K gene; therefore, vAbT33 can not grow on 
RK13 cells, which require the 29K gene product. Furthermore, vAbT33 forms 
blue plaques on permissive cells in the presence of the chromogenic 
substrate for betagalactosidase, Bluogal, due to the presence of the lacZ 
gene. See U.S. patent application Ser. No. 205,189, filed Jun. 10, 1988 
which corresponds to WO 89/12103. 
IVR vectors pAbT4592, pAbT4593, pAbT4597, pAbT4603, and pAbT621 were 
transfected into BSC-40 cells which had been infected with vaccinia virus 
vAbT33 (see Materials and Methods). Recombinant viruses were selected as 
white plaques in the presence of Bluogal on RK13 cells. Plaques were 
picked and purified, and were shown, by Southern analysis, to contain the 
appropriate SIVmac251 or HIV-1 gene(s): vAbT252 contains SIV gag-prot; 
vAbT271 contains HIV-1 env; vAbT253 contains SIV env; vAbT264 contains 
both SIV env and SIV gag-prot; and vAbT344 contains HIV-1 env and 
gag-prot. 
To insert SIVmac251 or HIV-1 genes into the vaccinia virus genome at the TK 
locus, which is located in the HindIII J region, a selection scheme based 
on the sensitivity of the TK.sup.+ viruses to bromodeoxyuridine (BUdR) 
was used. Bromodeoxyuridine is lethal for TK.sup.+ virus but allows 
recombinant, TK.sup.- virus to grow. Plasmids for in vivo recombination 
are therefore transfected into Hu143TK.sup.- cells which have been 
infected with a TK.sup.+ vaccinia virus (see Materials and Methods). 
In addition, plasmid vectors for insertion at TK contain the E. coli lacZ 
gene under the control of the vaccinia BamF promoter; recombinant viruses 
that contain the desired foreign gene(s) also contain the lacZ gene and 
therefore produce blue plaques when propagated in the presence of the 
chromogenic substrate for beta-galactosidase, Bluogal. Thus, recombinant 
viruses are selected in BUDR and are further identifiable as blue plaques 
in the presence of Bluogal. 
To construct a recombinant vaccinia virus that contains the HIV-1 gag gene 
inserted at the TK locus, the wild type NYCBH virus was used as the 
parental virus for in vivo recombination with vector pAbT164, to produce 
the recombinant virus vAbT141. 
To construct a recombinant vaccinia virus that contains the SIVmac251 env, 
gag-prot, and pol genes, vAbT264 was used as the parental virus for in 
vivo recombination with pAbT4583, an IVR vector that contains the 
SIVmac251 pol gene under the control of the vaccinia 40K promoter, flanked 
by DNA homologous to the vaccinia TK gene. This vector is described in 
U.S. patent application Ser. No. 205,454, filed Jun. 10, 1988 which 
corresponds to WO 89/12095. This yielded the recombinant virus vAbT277, 
which contains the SIVmac251 pol gene inserted at the TK locus under the 
control of the vaccinia 40K promoter, and the SIVmac251 env and gag-prot 
genes, under the control of the 40K and 30K vaccinia promoters, 
respectively, inserted at the HindIII M locus. 
EXAMPLE 4 
Black Plaque Assay for Expression of SIV or HIV-1 Antigens in Recombinant 
Vaccinia Virus. 
The black plaque assay, described in Materials and Methods, is an in situ 
enzyme-based immunoassay which can detect protein expressed by 
vaccinia-infected cells. This assay was performed on vaccinia recombinants 
vAbT252, vAbT253, vAbT264, and vAbT277 using serum from SIV-infected 
macaques, obtained from Ronald C. Desrosiers (New England Regional Primate 
Research Center, Southborough, Mass.), and on vAbT141 and vAbT344 using 
serum from HIV-1 infected human patients, obtained from John Sullivan 
(University of Massachusetts Medical School, Worcester, Mass.). 
Plaques formed by the negative control viruses NYCBH or vAbT33 showed only 
a background color which was consistent with the background on the cell 
monolayer itself. Plaques formed by vaccinia recombinants vAbT252, 
vAbT253, vAbT264, vAbT277, vAbT141, vAbT271 and vAbT344 stained a distinct 
dark purple color which was much darker than the background on the cell 
monolayer, showing that these recombinants strongly express SIVmac251 or 
HIV-1 antigens. 
EXAMPLE 5 
Immunoprecipitation of SIVmac251 or HIV-1 Antigens from Recombinant 
Vaccinia Viruses. 
Immunoprecipitation analysis was performed on cells infected with 
recombinant vaccinia viruses vAbT252, vAbT253, vAbT223, vAbT264, vAbT277, 
vAbT141, vAbT271 and vAbT344 as described in Materials and Methods. The 
results, which are summarized in Table 1, show that each of these vaccinia 
recombinants expresses the encoded polypeptide(s). 
TABLE 1 
______________________________________ 
Immunoprecipitation of SIVmac251 or HIV-1 
polypeptides from recombinant vaccinia viruses 
Vaccinia recombinant 
Inserted genes 
Proteins observed 
______________________________________ 
vAbT252 SIV gag-prot p55, p40, p24, p15 
vAbT253 SIV env gp160, gp120, gp32 
vAbT264 SIV env, gag-prot 
gp160, gp120, gp32 
p55, p40, p24, p15 
vAbT277 SIV env, gag-prot, 
gp160, gp120, gp32 
pol p55, p40, p24, p15 
p64, p53, p10 
vAbT141 HIV gag p55 
vAbT271 HIV env gp160, gp120, gp41 
vAbT344 HIV env, gag-prot 
gp160, gp120, gp41 
p55, p40, p24, p17 
______________________________________ 
EXAMPLE 6 
Biochemical Detection of Retroviral Particles Produced by Vaccinia 
Recombinants that Express SIV or HIV Antigens. 
Expression analysis described in Example 5 can be used to confirm the 
synthesis of the polypeptides encoded by inserted HIV or SIV genes, but 
does not address the question of whether these polypeptides self-assemble 
in vivo into defective viral particles. As one means of determining 
whether vaccinia recombinants that express env, gag-prot or both env and 
gag-prot produce retroviral-like particles released into the medium of 
infected cells, the medium was examined for the presence of structures 
containing env and/or gag polypeptides which could be pelleted by 
centrifugation. BSC-40 cells were infected with SIV/vaccinia recombinants 
vAbT253 (env), vAbT252 (gag-prot) or vAbT264 (env and gag-prot) or with 
HIV/vaccinia recombinants vAbT141 (gag), vAbT271 (env) or vAbT344 (env and 
gag-prot). Infected cells were labeled with .sup.35 S! methionine as 
described in Materials and Methods. After 16-18 hours of infection, the 
medium was collected and clarified by centrifugation at 1000 rpm for 5 
minutes. The resulting supernatant was removed and subjected to 
centrifugation at 24K for 90 minutes in an 8W28 rotor. The supernatant was 
then removed and the resulting pellet was resuspended in 1 ml PBS buffer 
(13 mM NaCl, 2.7 mM KCl, 8.1 mM Na.sub.2 HPO.sub.4, 1.5 mM KH.sub.2 
PO.sub.4). Samples from the supernatant and pellet were subjected to 
immunoprecipitation analysis, using macaque anti-SIV antiserum, for 
SIV/vaccinia recombinants, or human anti-HIV antiserum, for HIV/vaccinia 
recombinants, as described in Materials and Methods. The results showed 
that for env and gag-prot recombinants vAbT264 and vAbT344, while the 
supernatant contained only gp120, which had been presumably shed into the 
culture medium during growth of the SIV/vaccinia recombinant (Kieny et 
al., 1986. Bio/Technology 4:790), the pellet contained not only gp120, but 
also the env gene-encoded gp32 (for vAbT264) or gp41 (for vAbT344) as well 
as the gag gene-encoded p55, p40, p24, and p15. These results strongly 
suggested that the recombinant vaccinia-produced env and gag proteins 
self-assemble into particles or complexes. 
For vAbT141 and vAbT252, which express only the HIV gag or SIV gag-prot 
proteins, respectively, pelleted material contained gag proteins, 
consistent with the formation of immature viral particles by self-assembly 
of gag polypeptides. By contrast, for vAbT253 and vAbT271 which express 
only the SIV or HIV env glycoproteins, respectively, no immunoprecipitable 
polypeptides were found in the pellet, although substantial amounts of 
gp120 were found in the supernatant. These results were consistent with 
the prediction that defective viral particle formation occurs only when 
the gag polypeptides are expressed, alone or in combination with other 
viral structural proteins such as the env glycoproteins. 
EXAMPLE 7 
Demonstration of Retroviral Particle Formation by Recombinant Vaccinia 
Viruses that Express SIV or HIV-1 Polypeptides using Electron Microscopy 
As an additional confirmation that the recombinant vaccinia viruses that 
express gag, gag+env, or gag+env+pol polypeptides are capable of 
assembling into retroviral-like particles, lysate of cells infected with 
these recombinant vaccinia viruses were visually examined by electron 
microscopy by the methods detailed in the Materials and Methods section 
above. 
These experiments showed that vaccinia recombinants that express gag, 
(vAbT141 or vAbT252) env+gag, (vAbT223, vAbT264 or vAbT344) or env+gag+pol 
(vAbT277) genes give rise to the formation of enveloped retroviral 
particles; in the recombinants that coexpress gag and env polypeptides, 
(vAbT223 vAbT264, vAbT344 and vAbT277) the particle envelope contains the 
envelope glycoproteins, as shown by the presence of glycoprotein "spikes" 
on the surface of the particles. For the SIV recombinant vAbT223, the 
presence of SIV envelope glycoproteins on the surface of the particles was 
also demonstrated with immunogold electron microscopy, using a monoclonal 
antibody directed against one of the envelope glycoproteins. 
Plasmid Deposits 
The plasmids E. coli MC1061 pAbT 4597 and E. coli MC 1061 pAbT621 were 
placed on deposit, under provisions of the Budapest Treaty, at the 
American Type Culture Collection in Rockville, Md. on May 31, 1989. The 
plasmids have been assigned the accession numbers 67998 and 67999, 
respectively. 
Equivalents 
Those skilled in the art will recognize, or be able to ascertain using no 
more than routine experimentation, many equivalents to the specific 
embodiments of the invention described herein. Such equivalents are 
intended to be encompassed by the following claims.