JP2003509013A - Stabilized viral envelope proteins and uses thereof - Google Patents

Abstract

  (57) [Summary] The present invention provides an isolated nucleic acid comprising a nucleotide segment having a sequence encoding a viral envelope protein comprising a viral surface protein and a corresponding viral transmembrane protein, wherein the viral envelope protein comprises the viral surface protein. A nucleic acid containing one or more mutations in the amino acid sequence that increases the stability of the complex formed between the protein and the transmembrane protein. The present invention relates to a virus envelope protein comprising a virus surface protein and a corresponding transmembrane protein, wherein the virus envelope protein has a stability of a complex formed between the virus surface protein and the transmembrane protein. Virus envelope proteins containing one or more mutations in the amino acid sequence that increase The invention further provides a method of treating an HIV-1 infection.

Description

Detailed Description of the Invention

This application is based on US application Ser. No. 09 / 340,99 filed June 25, 1999.
No. 2 is a continuation-in-part application, claiming its priority, and its contents are
Incorporated herein by reference.

The invention disclosed herein is directed to NIH Grant No. R01 from the Ministry of Health and Human Services.
It was made with the assistance of the government by Al45463-01. Therefore, the government also has certain rights in the invention.

Throughout this application, various publications are referenced. The disclosure content of these references is
In order to more fully describe the technical field to which the present invention pertains, the present application is incorporated by reference.

[0004]

BACKGROUND OF THE INVENTION

Human immunodeficiency virus (HIV) is the pathogen that causes AIDS, a fatal disease characterized by a compromised function of the immune system. In the early phase of the HIV replication cycle, fusion of the virus with the cell membrane occurs after the virus attaches to the host cells that are sensitive to the virus. These phenomena are mediated by the outer viral envelope glycoprotein, which is first synthesized as a fusion-deficient precursor envelope glycoprotein (env) known as gp160. The gp160 glycoprotein is endoproteolytic.
, the mature envelope glycoproteins gp120 and g
processed to p41 (these glycoproteins are non-covalently associated on the surface of the virus). The gp120 surface protein has a high affinity binding site for human CD4, which is the primary receptor for HIV, and chemokine receptor CCR.
5 and domains that interact with fusion coreceptors such as CXCR4. The gp41 protein spans the viral membrane and contains at its amino terminus an amino acid sequence important for fusion of the viral and cell membranes. HIV envelope glycoproteins form non-covalently bound oligomers of gp120 / gp41 (often trimers) on the surface of viruses. Although the detailed mechanism of virus entry is not yet fully understood, gp41-mediated virus-cell fusion following gp120 first binds to CD4 and subsequently to a chemokine receptor that induces fusion Happens.

The HIV envelope glycoprotein, located on the surface of the virion and plays a central role in mediating viral entry, represents an important goal in the development of HIV vaccines. Although many HIV-infected individuals show strong antibody (Ab) responses to the envelope glycoprotein, anti-gp12 produced during spontaneous infection
Many 0 and anti-gp41 antibodies bind weakly or not at all to virions and are therefore not functionally effective. These antibodies are probably raised against "viral debris" containing gp120 monomers released from virions or infected cells or improperly processed oligomers,
Affinity for them matures (Burton and Montefior
i, AIDS, 11 [Suppl A]: 587, 1997).

Several prophylactic HIV-1 subunit vaccines have been tested in Phase I and II clinical trials and multivalent formulations have entered Phase III trials. These vaccines are available in the form of monomeric gp120 or unprocessed gp16.
It contains any of 0 proteins. In addition, many of the vaccines are derived from viruses that have been adapted to grow to high levels in immortalized T cell lines (TCLA virus). These vaccines consistently elicit antibodies that neutralize homologous strains of the virus and also some TCLA viruses. However, the antibody does not strongly neutralize primary HIV-1 isolates (Mascola et al.
al. J. INFEC. Dis. 173: 340, 1996). Compared to TCLA strains, clinically more relevant primary isolates typically have different cytotropism, exhibit different patterns of coreceptor usage, and are more susceptible to neutralization by soluble CD4 and antibodies. is decreasing. These differences are primarily located in the viral envelope glycoprotein (Moore and Ho, AIDS, 9).
[Suppl A]: S117-S136, 1995).

Importance of Oligomerization in Envelope Glycoprotein Structures It has been recognized that current generation HIV subunit vaccines do not adequately present key neutralizing epitopes when they appear on virions (Parre).
n et al, Nat. Med. 3: 366, 1997). The natural structure of virions affects the presentation of antibody epitopes in several ways. Primarily,
Much of the surface area of gp120 and gp41 is blocked by interactions between subunits in the trimer. Therefore, some regions of gp120 that are well exposed (and therefore highly immunogenic) on the monomeric form of the protein, especially around the N- and C-termini, are also intact on the native trimer. Becomes inaccessible (Moor
e et al. Virol 68: 469, 1994). This may occur during natural infection (due to efflux of gp120 monomers from virions or infected cells) or after vaccination with gp120 subunits.
This means that the subset of antibodies raised against 120 monomers is not suitable. This provides another level of protection against the virus. The immune system is less reactive and therefore erroneously makes antibodies to shed gp120 that are not effective against virions.

Second, there is a more subtle problem in that the structure of the key gp120 epitope can be affected by oligomerization. One classic example is given by the epitope for human MAb IgG1b12, which has extensive neutralizing capacity (Bu
rton et al. Science 266: 1024, 1994). This epitope overlaps with the CD4 binding site on gp120 and results in monomeric gp12.
Exists above 0. However, IgG1b12 reacts with the native oligomeric gp120 much more than could be expected from its monomeric reactivity, which explains its unusually strong neutralizing activity (77, 99-103). Therefore, the IgG1b1
The two epitopes are oligomer-dependent but not oligomer-specific. Unfortunately, the opposite situation is more common. That is, many antibodies do not neutralize the virus because they react strongly with the epitope for the CD4 binding site on monomeric gp120 but not with the native trimer. By some unclear manner, oligomerization of gp120 is adversely affected by the structure recognized by these MAbs (Fouts et al., J Virol.
71: 2779, 1997).

A third example of the problem caused by the native structure of HIV-1 envelope glycoprotein is given by the gp41 MAb. Only one gp41 MAb (2F5) is known to have strong neutralizing activity against the primary virus.
Only (Trkola et al., J Virol, 69: 6609,
1995), among the tested, only 2F5 is intact gp120-.
It is believed to recognize the gp41 complex (Satentau et al.
al, Virology 206: 713, 1995). Possibly all other gp41 MAbs that bind to virions or virus infected cells are gp120
Reacts with the dissociated gp41 structure lacking the fusion ability. The most stable form of gp41 is due to this post-fusion configuration (Weissenhorn et al, N.
Aure, 387: 426, 1997), many anti-gp41 antibodies have been shown to react against inappropriate gp41 structures that do not exist on pre-fusion forms (during natural infection or g.
It can be assumed that it is produced (after vaccination with p160).

Despite these defense mechanisms, many HIV-1 isolates are strongly neutralized by a limited group of broadly reactive human monoclonal antibodies (MAbs), thus inducing an appropriate humoral immune response. It is not impossible to do. Mab IgG1b12 is gp1
Another monoclonal antibody (2G12; Trko
la et al. J Virol 70: 1100, 1996) is mostly due to steric hindrance of virus-cell attachment; 2F5 acts by directly impairing the fusion reaction itself. To understand the neutralizing capacity of these MAbs, they react preferentially with the fusogenic oligomeric forms of the envelope glycoprotein (as found on the surface of cells infected with virions and viruses). Is important (Parren et al J. Viro
72: 3512, 1998). These monoclonal antibodies are thereby distinguished from the less active ones. The limited number of MAbs that react with oligomers explains why so few MAbs can neutralize the primary virus. Thus, with rare exceptions, neutralizing anti-HIV antibodies can bind infectious virus, but non-neutralizing antibodies cannot (Fout.
s et al AIDS Res Human Retrovir. 14: 5
91, 1998). Neutralizing antibodies also have the ability to clear infectious virus through effector functions such as complement-mediated viral membrane lysis.

Modification of the HIV Envelope Glycoprotein Antigenic Structure HIV has evolved sophisticated mechanisms to shield key neutralization sites from the humoral immune response, and, in principle, these mechanisms are: It can be "useless" in a vaccine. One example is the V3 loop, which is the major antigenic epitope that induces the antibody response to diverge from the more widely conserved neutralizing epitope, especially in the case of the TCLA virus. HIV-1 is also protected from humoral immunity by extensive glycosylation of gp120. V1 of SIV gp120
Deletion of the glycosylation site from the / V2 loop not only produced a virus that was sensitive to neutralization, but also increased the immunogenicity of the mutant virus, resulting in a stronger immune response to the wild-type virus. Happened (Reitter et al,
Nat Med 4: 679, 1998). Similarly, removal of the V1 / V2 loop from HIV-1 gp120 leaves the conserved region underneath vulnerable to antibody attack (Cao et al, J. Virol. 71: 9808, 1997).
), Whether it improves immunogenicity is still unclear.

Notably, the envelope glycoprotein V1 of the TCLA virus,
Deletion of the V2 and V3 loops did not improve the induction of neutralizing antibodies in the DNA vaccine (Lu et al, AIDS Res Human Re.
trovir 14: 151, 1998). However, gp120 and gp4
1 interaction instability (probably exacerbated by deletion of the variable loop)
It may affect the results of this experiment. Thus, the stabilization of gp120-gp41 described in the present invention may be useful for DNA plasmids, viral vectors, and other nucleic acid-based HIV vaccines. gp120-
By increasing the time that the gp41 complex is presented to the immune system, the stabilized envelope protein expressed in vivo provides, in principle, a means to significantly improve the immune response induced during natural infection.

Native and non-natural oligomeric forms of the HIV envelope glycoprotein Current data indicate that the three gp120 moieties are non-covalently associated with the three underlying gp41 components in a metastable configuration. (The fusion ability is
It is caused by the interaction with cell surface receptors). This pre-fusion form may optimally present neutralizing epitopes. We refer to this form of the envelope glycoprotein as native gp120-gp41. However, other oligomeric forms are possible and it is important to establish these (see Figure 1).

Gp160 : Full-length gp160 molecules contain a hydrophobic transmembrane domain so that they often aggregate when expressed at least in part as a recombinant protein. One such molecule is the gp120 / gp41 precursor of the gp160 precursor.
Derived from a naturally occurring mutation that prevents processing into the (VanCott et a
l J Virol 71: 4319, 1997). The gp160 precursor is
It does not mediate virus-cell fusion and is insufficient as a mimicry of gp120 / gp41 having a fusion ability. Recombinant gp160 molecules had no advantage over gp120 monomers when evaluated in humans (Gorse et al., Vac.
cine 16: 493, 1998).

Uncleaved gp140 (gp140UNC) : A stable "oligomer" is the above-mentioned gp
It has been created by removing the natural proteolytic site necessary to convert the 160 precursor protein into gp120 and gp41 (Berman et al.
al. , J Virol. 63: 3489, 1989; Earl et al.
． , Proc. Natl Acad Sci 87: 648, 1990). In order to express these constructs as soluble proteins, a stop codon is inserted within the env gene to truncate the protein just before the transmembrane segment of gp41. The protein lacks the transmembrane domain of gp41 and the long cytoplasmic tail, but retains regions important for viral entry and induction of neutralizing antibodies. The secreted protein contains full-length gp120 covalently linked to the ectodomain of gp41 through a peptide bond. The protein is SDS-PA
It migrates in GE under both reducing and non-reducing conditions as a single species with an apparent molecular weight of approximately 140 kilodaltons (kDa). The protein probably forms higher molecular weight non-covalently linked oligomers through interactions mediated by the gp41 moiety.

[0016] Several lines of evidence indicate that the uncleaved gp140 molecule is the native gp120-g
It has been suggested that it does not adopt the same arrangement as p41. These include evidence from the observations described herein and the finding that the uncleaved gp140-gp41 complex does not positively bind fusion coreceptors (R. Doms, personal communication). Furthermore, this type of gp140 protein failed to efficiently select neutralizing MAbs when used to probe phage display libraries, whereas virions were efficient (Parren et al., J Virol.
70: 9046, 1996). The present inventors have found that the uncleaved gp120-gp41
The ectodomain material is called gp140UNC.

Cleavable but uncleaved gp140 (gp140NON) : During biosynthesis, gp160 is cleaved by the furin family of cellular endoproteases into gp120 and gp41. As shown below, gp41
The ability of mammalian cells to cleave from gp120 is limited. Therefore, when the envelope glycoprotein is overexpressed, the envelope glycoprotein can saturate the endogenous furin enzyme and be secreted in the form of a precursor. Since these molecules may be cleavable, we have defined them as gp140NON.
Called. Like gp140UNC, gp140NON is SDS-PAGE
In, it migrates with an apparent molecular weight of approximately 140 kilodaltons (kDa) under both reducing and non-reducing conditions. As shown below, gp140NON
It seems to have the same unnatural topology as 40UNC.

Cleaved gp140 (gp140CUT) : gp140CUT refers to the fully processed ectodomain gp41 with the full length gp120 which is fully processed and capable of forming oligomers as found on virions. The non-covalent interaction between gp120 and gp41 has a long lifespan that allows the virus to associate with new target cells and initiate fusion (complete with natural infection within minutes). This is a possible process). However, at present, this association has been shown to be unstable to produce substantial amounts of truncated gp140 in a nearly homogeneous form.

Stabilization of Viral Envelope Glycoproteins The metastable pre-fusion conformation of viral envelope proteins such as gp120 / gp41 is stable enough to keep the infection spreading but is required for virus-cell fusion. It is unstable enough to easily give rise to various conformational changes. In the HIV isolates examined thus far, the gp120-g
The p41 interaction has been shown to be too unstable to produce gp140CUT as a secreted protein on a preparative scale. However, due to the vast genetic diversity of HIV, it is believed that screening methods such as those described herein may be used to identify viruses with excellent env stability. Alternatively, in principle, it may be possible to select the virus with enhanced stability after continuously placing the virus under conditions known to destabilize the gp120-gp41 interaction. Such conditions include 37-6
Increasing temperature in the range of 0 ° C. and / or low concentrations of surfactants or chaotropic agents may be included. Envelope proteins from such viruses can also be subcloned into the pPPI4 expression vector and analyzed for stability using our method.

Semi-empirical modification approaches for stabilizing viral envelope proteins can also be employed. For example, stable heterodimers have been successfully created by introducing complementary "knob" and "hole" mutations in the binding partner (Atwell et al., J. Mol. Biol. 4: 26,199
7). Alternatively or in addition, other favorable interactions such as salt bridges, hydrogen bonds, or hydrophobic interactions can be introduced. This approach is
And facilitated by increasing the understanding of the structure of TM proteins, and the results described herein contribute to such understanding.

As demonstrated in the present invention, stabilization of SU-TM may also be achieved by the introduction of one or more disulfide bonds. Among the mammalian retroviruses,
Only lentiviruses like HIV have a non-covalent association between the surface (SU) and transmembrane (TM) glycoproteins. In contrast, type C and type D retroviruses all have disulfide bonds between subunits. The ectodomain of the retroviral TM glycoprotein has a broadly common structure,
One universal feature is the presence of a small Cys-Cys binding loop approximately in the center of the ectodomain. In the TM glycoproteins of type C and D retroviruses, an unpaired cysteine residue is found immediately C-terminal to this loop and is almost certainly used to form the SU-TM disulfide bond. (Gal
laher et al. , AIDS Res Human Retrovir
11: 191, 1995; Schultz et al. , AIDS Res
Human Retrotro 8: 1585, 1992).

The gp41 and other lentiviral TM glycoproteins lack the third cysteine, but structural homology suggests that one may be inserted near the short central loop structure. Thus, there is strong evidence based on mutations indicating that the first and last conserved regions of gp120 (C1 and C5 domains) may be contact sites for gp41.

The present invention provides isolated, nucleic acid molecules that encode stabilized, antigenically standard forms of mutant viral surfaces and mutant viral proteins. The present invention describes the design and synthesis of the stabilized viral proteins. Importantly, using appropriate methods to influence the stabilization, the viral proteins adopt a conformation with the desired characteristics. The invention further provides vaccines based on proteins or nucleic acids containing mutant viral envelope proteins, antibodies isolated or identified using mutant viral envelope proteins, pharmaceutical compositions containing these vaccines or antibodies, and HIV. The use of these compositions for treating or preventing infections of viruses such as The present invention describes the use of said mutant viral proteins to identify if a compound may inhibit the virus, and the compounds thus identified.

[0024]

[Outline of the Invention]

The present invention is an isolated nucleic acid comprising a nucleotide segment having a sequence encoding a viral envelope protein comprising a viral surface protein and a corresponding viral transmembrane protein, wherein the viral envelope protein is the viral surface protein. Provides an isolated nucleic acid containing in the amino acid sequence one or more mutations that increase the stability of the complex formed between and the viral transmembrane protein.

The present invention provides a mutant viral envelope protein that differs in at least one amino acid from the sequence of the corresponding wild-type viral envelope protein, comprising:
A nucleotide having a sequence encoding a mutant viral envelope protein, which has increased stability as compared to the corresponding complex obtained from the wild-type envelope protein and gives a complex containing a surface protein and a transmembrane protein during proteolysis. An isolated nucleic acid comprising a segment is provided.

In one embodiment of the above, the viral surface protein is HIV-1 gp12.
0 or a modified gp120 whose immunogenicity is modified as compared to wild-type gp120. In one aspect, the transmembrane protein is a modified gp41 that is immunogenically modified as compared to HIV-1 gp41 or wild-type gp41.

The present invention provides a vaccine comprising the above isolated nucleic acid. In one aspect, the vaccine comprises a therapeutically effective amount of the nucleic acid. In another aspect, the vaccine comprises a therapeutically effective amount of the protein encoded by the nucleic acid. In another aspect,
The vaccine comprises a combination of the recombinant nucleic acid molecule and the mutant viral envelope protein.

The present invention provides a method of treating a viral disease, comprising treating said patient by immunizing a patient infected with the virus using the above vaccine or a combination thereof.

[0029]   The invention provides a vaccine comprising a prophylactically effective amount of the above isolated nucleic acid.

The present invention provides a vaccine comprising a prophylactically effective amount of a protein encoded by the above nucleic acid.

The present invention provides a method of reducing the likelihood of a subject being infected with a virus, comprising administering the vaccine or a combination thereof to reduce the subject's likelihood of becoming infected with the virus. Provide a method.

The present invention comprises: a recombinant subunit protein, a DNA plasmid,
There is provided the above vaccine comprising, but not limited to, an RNA molecule, a replicating viral vector, a non-replicating viral vector, or a combination thereof.

The present invention is a method of reducing the severity of a viral disease in a patient, wherein the patient is exposed to the virus prior to exposure to the virus by administering the above vaccine or a combination thereof. Sometimes, a method is provided that comprises reducing the severity of a viral disease in a patient.

The present invention is a mutant viral envelope protein that differs by at least one amino acid from the sequence of the corresponding wild-type protein, and has increased stability over the corresponding complex obtained from the wild-type envelope protein. And a mutant virus envelope protein which provides a complex comprising a surface protein and a transmembrane protein during proteolysis.

The present invention is a complex comprising a viral surface protein and a viral transmembrane protein, which has increased stability as compared to the corresponding complex obtained from the wild type envelope protein, At least one wild-type protein sequence
Provided is a complex obtained by proteolytic cleavage of a mutant viral envelope protein having different sequences of three amino acids.

The present invention provides a mutant viral envelope protein encoded by the above nucleic acid molecule.

The present invention provides a vaccine comprising a therapeutically effective amount of the above protein or complex. The invention also provides a vaccine comprising a prophylactically effective amount of the above protein or complex.

The present invention provides a method of stimulating or enhancing the production of an antibody that recognizes the above protein or complex in a subject.

The present invention provides a method of stimulating or enhancing the production of cytotoxic T lymphocytes that recognize the above proteins in a subject.

The present invention provides an antibody capable of specifically binding to the above mutant protein. The present invention also provides an antibody that can specifically bind to the above mutant protein or complex, but does not bind to the wild-type protein or complex.

The present invention provides an antibody, antibody chain or fragment thereof identified using a viral envelope protein encoded by the above recombinant nucleic acid molecule. The antibody may be of the IgM, IgA, IgE, or IgG class or subclasses thereof. The above antibody fragments include, but are not limited to, Fab, Fab ′, (Fab ′) ₂ , Fv and single chain antibodies.

The present invention provides an isolated antibody light chain of the above antibody, or a fragment or oligomer thereof. The present invention also provides an isolated antibody heavy chain of the above antibodies, or fragments or oligomers thereof. The invention also provides one or more CDR regions of the above antibodies. In one aspect, the antibody is derivatized. In another aspect, the antibody is a human antibody. The antibody includes, but is not limited to, a monoclonal antibody and a polyclonal antibody. In some embodiments, the antibody is humanized.

[0043]   The present invention provides an isolated nucleic acid molecule encoding the above antibody.

The present invention is a method of reducing the likelihood of viral infection of a subject exposed to the virus, wherein said subject is infected with the virus by administering said antibody or said isolated nucleic acid. A method comprising reducing the likelihood is provided. In a preferred embodiment, the virus is HIV.

The present invention provides a method of treating a patient infected with a virus, the method comprising treating said patient by administering said antibody or said isolated nucleic acid. In a preferred embodiment, the virus is HIV.

The present invention provides a substance capable of binding the mutant viral envelope protein encoded by the above recombinant nucleic acid molecule. In one aspect, the substance inhibits a viral infection.

The present invention provides a method for determining whether a compound can inhibit viral infection, which comprises (A) a mutant viral envelope protein encoded by the recombinant nucleic acid of claim 1 wherein: Contacting an appropriate concentration of the compound under conditions that allow binding to the protein; (B) allowing the resulting complex to bind to the mutant virus envelope protein of the reporter molecule. Contacting with a reporter molecule; (C) measuring the amount of bound reporter molecule; (D) measuring the amount of bound reporter molecule in step (C) in the absence of said compound Comparing, and the decrease in amount is an indicator that the compound can inhibit infection by a virus, so that To provide a method for determining whether or not capable of inhibiting the scan infection.

The present invention provides a method for determining whether a compound can inhibit viral infection, comprising: (a) a host cell viral receptor or a molecular mimic thereof, wherein said compound and receptor or receptor Contacting a suitable concentration of said compound under conditions which allow the binding of a mimetic; (b) in the absence of said compound in the mutant viral envelope protein encoded by the recombinant nucleic acid of claim 1. Contacting the resulting complex under conditions that allow the binding of said envelope protein to a receptor or receptor mimic in (c) the amount of binding of the envelope protein to the receptor or receptor mimic Measuring; and (d) comparing the amount of binding determined in step (c) with the amount measured in the absence of said compound, wherein By the indication that may inhibit infection by virus, it provides a method for determining whether a compound is capable of inhibiting viral infection.

The present invention further provides a simple method for determining whether a subject has produced antibodies capable of blocking viral infectivity.

The present invention provides the above method, wherein said compound is a previously unknown compound.

The present invention provides a compound determined to be capable of inhibiting viral infection by the above method.

The present invention provides a pharmaceutical composition comprising an amount of a compound effective to inhibit a viral infection determined by the above method that is capable of inhibiting a viral infection and a pharmaceutically acceptable carrier. provide. In one aspect, the viral infection is an HIV-1 infection. In a preferred embodiment, the virus is HIV.

The present invention differs from the corresponding wild-type protein by at least one amino acid, has increased stability over the corresponding complex obtained from the wild-type envelope protein, and is associated with surface proteins and transmembranes. A mutant virus envelope protein that provides a complex containing a protein, wherein the surface protein and the transmembrane protein are encoded by different nucleic acids.

The present invention comprises a viral surface protein and a transmembrane protein, has increased stability over the corresponding complex obtained from the wild-type envelope protein, and the sequence of the corresponding wild-type protein is A complex provided by proteolysis of a mutant viral envelope protein having a sequence differing in at least one amino acid, wherein the surface protein and the transmembrane protein are encoded by different nucleic acids.

The present invention provides an antibody that binds to the above protein or complex but does not cross-react with individual monomeric surface proteins or individual monomeric transmembrane proteins. The present invention provides the above antibody capable of binding to HIV-1 virus.

[0056]

Detailed Description of the Invention

The present invention is an isolated nucleic acid comprising a nucleotide segment having a sequence encoding a viral envelope protein comprising a viral surface protein and a corresponding viral transmembrane protein, wherein the viral envelope protein is the viral surface protein. Provides an isolated nucleic acid containing in the amino acid sequence one or more mutations that increase the stability of the complex formed between and the viral transmembrane protein.

The present invention provides mutant viral envelope proteins that differ in at least one amino acid from the sequence of the corresponding wild-type viral envelope protein,
A sequence encoding a mutant viral envelope protein, which by proteolysis, provides a complex with a surface protein and a transmembrane protein with increased stability as compared to the corresponding complex obtained from the wild type envelope protein. An isolated nucleic acid comprising a nucleotide segment having is provided.

As used herein, “increasing stability” means longer life,
That is, it means that it is difficult to dissociate. The interaction may be stabilized by introducing disulfide bonds, salt bridges, hydrogen bonds, hydrophobic interactions, forward van der Waals contacts, linker peptides, or combinations thereof. The stabilizing interaction may be introduced by recombinant methods. Alternatively, or in addition to this, after placing the virus under conditions known to destabilize the interaction between the surface protein and the transmembrane envelope protein, a selection method such as selecting a resistant virus is used. , A stabilized viral envelope protein may be obtained. Until a viral envelope protein with the desired stability is obtained,
This step may be repeated once or more. Alternatively, one screens for isolates with naturally occurring mutations that increase the stability of the interaction between the surface and transmembrane proteins compared to the stability observed with the original wild-type viral envelope protein. May be.

The present invention provides for the expression of the precursor envelope protein in the absence of a sufficient amount of endoprotease, or by mutating the endoproteolytic cleavage site in the absence of additional mutations ( It does not include known viral proteins that interfere with endoproteolytic processing of precursor envelope proteins that separate surface and transmembrane proteins).
In such known viral envelope proteins, the viral surface and transmembrane proteins are physically bound by a covalent bond, but it is not known to form a complex as shown in FIG. .

One embodiment of the above virus is a lentivirus. In one aspect, the virus is simian immunodeficiency virus. Another aspect of the virus is the human immunodeficiency virus (HIV). Said virus is known as two known species of HIV (HIV
-1 or HIV-2). The HIV-1 virus can be of any of the major known subtypes (Clades A, B, C, D, E, F, G, and H) or minor minor subtypes (Group O). Further discovery of HIV type, subtype, or class may be used in the present invention.
In one aspect, the human immunodeficiency virus is a primary isolate. In one aspect, the human immunodeficiency virus is HIV-1 _JR-FL . In another aspect, the human immunodeficiency virus is HIV-1 _DH123 . In another aspect, the human immunodeficiency virus is HIV-1 _Gun-1 . In another aspect, the human immunodeficiency virus is HIV-1 _89.6 . In another aspect, the human immunodeficiency virus is HIV-1 _HXB2 .

HIV-1 _JR-FL is the first strain isolated from brain tissue of an AIDS patient collected by autopsy and cultured with lectin-activated normal human PBMC (O'Brien et al. , Nature, 348: 69, 199.
0). HIV-1 _JR-FL can replicate in phytohemagglutinin (PHA) stimulated PBMCs and blood-derived macrophages using CCR5 as a fusion co-receptor, but in many immortalized T cell lines. It is known that they do not replicate efficiently.

HIV-1 _DH123 is the first viral clone isolated from peripheral mononuclear cells (PBMC) of AIDS patients (Shibata et al., J. Chem.
Virol 69: 4453, 1995). HIV-1 _DH123 uses both CCR5 and CXCR4 as fusion _co -receptors and PHA-stimulated PBM
It is known to be capable of replicating in C, blood-derived macrophages, and immortalized T cell lines.

HIV-1 _Gun-1 is the first cloned virus isolated from peripheral blood mononuclear cells of hemophilia B patients with AIDS (Takeuchi et al.
． , Jpn J Cancer Res 78:11 1987). HIV-
1 _Gun-1 uses both CCR5 and CXCR4 as fusion _co- receptors,
It is known to be able to replicate in PHA-stimulated PBMCs, blood-derived macrophages, and immortalized T cell lines.

HIV-1 _89.6 is the first cloned virus to be isolated from AIDS patients (Collman et al, J. Virol. 66:75).
17, 1992). It is known that HIV-1 _89.6 uses both CCR5 and CXCR4 as fusion _co -receptors and can replicate in PHA-stimulated PBMCs, blood-derived macrophages, and immortalized T cell lines. ing.

HIV-1 _HXB2 can replicate in PHA-stimulated PBMCs and immortalized T cell lines, but not in blood-derived macrophages, using CXCR4 as a fusion _coreceptor. It is a known TCLA virus.

The above strains are used to make the mutant viral envelope proteins of the present invention, but instead of them, as is well known to those skilled in the art, other HIV-
It is also possible to substitute one strain.

Some embodiments of the above viral surface proteins include gp120, or wild type gp12.
It is a modified version of gp120 with altered immunogenicity compared to 0. In one aspect, the modified gp120 molecule is characterized by the absence of one or more variable loops present in wild-type gp120. In one aspect, the variable loop is V1.
, V2, or V3. In one aspect, the modified gp120 molecule is characterized by the absence or presence of one or more canonical glycosylation sites not present in wild-type gp120. In some embodiments, one or more canonical glycosylation sites are
It is not present in the V1V2 region of the gp120 molecule.

In one aspect, the transmembrane protein is gp41, or a modified form of gp41 having altered immunogenicity relative to wild-type gp41. In one aspect, the transmembrane protein is full length gp41. In another aspect, the transmembrane protein contains an ectodomain of gp41 and a membrane anchoring sequence, but lacks some or all of the cytoplasmic sequence of gp41. In one aspect, the transmembrane protein is gp
41 External domain. In one aspect, the transmembrane protein is modified by the deletion or insertion of one or more canonical glycosylation sites.

One embodiment of the above virus surface protein is gp120 or a derivative thereof. In some embodiments, the gp120 molecule is modified by the deletion or truncation of one or more variable loop sequences. The variable loop sequences include, but are not limited to, V1, V2, V3, or combinations thereof. In another aspect, the gp120 molecule is modified by the deletion or insertion of one or more standard glycosylation sites. In the region of gp120 where the canonical glycosylation site is deleted, gp120
Includes, but is not limited to, the V1V2 region of the molecule.

The V1, V2, and V3 variable loop sequences of HIV-1 _JR-FL are shown in FIG. The amino acid sequences in these variable loops, although likely different in other HIV isolates, will be located in the homologous region of the gp120 envelope glycoprotein.

As used herein, “standard canonical glycosylation sites (canonical glyc
"Assylation site)" refers to the Asn that provides the N-binding site for the
-X-Ser, or Asn-X-Thr amino acid sequences are included, but are not limited to. Furthermore, Ser or Thr residues capable of linking carbohydrates via O-bonds that are not present in such sequences are also “standard glycosylation sites”. In the latter "canonical glycosylation site," the O-linked glycosylation site will be removed by mutating the Ser and Thr residues to amino acids other than serine or threonine.

“Derivatives” as used with respect to gp41 removes or masks the gp41 ectodomain, gp41 modified by the deletion or insertion of one or more glycosylation sites, the well known major antigen epitopes. Modified gp41, gp41 fusion proteins, and gp41 labeled with an affinity ligand or other detectable marker,
It is not limited to these.

As used herein, the “ectodomain” means the extracellular region or a part thereof excluding the transmembrane region and the cytoplasmic region.

In some embodiments, stabilization of the mutant viral envelope protein is achieved by introducing one or more cysteine-cysteine bonds between the surface protein and the transmembrane protein.

In some embodiments, one or more amino acids adjacent to or containing atoms within 5 Å of the introduced cysteine are mutated to non-cysteine residues.

As used herein, “adjacent” means immediately before or after the primary sequence of a protein.

As used herein, “mutated” means different from wild type.

As used herein, “non-cysteine residue” means an amino acid other than cysteine.

In some embodiments, one or more cysteines present in gp120 or modified gp120 are disulfide linked to one or more cysteines in gp41 or modified gp41.

In some embodiments, the cysteine in the C5 region of gp120 or modified gp120 is disulfide linked to a cysteine in gp41 or the modified ectodomain. In some embodiments, a disulfide bond is formed between the cysteine introduced by the A492C mutation in gp120 or modified gp120 and the cysteine introduced by the T596C mutation in gp41 or modified gp41.

By “C5 region” as used herein is meant the fifth conserved amino acid sequence present in the gp120 glycoprotein. The C5 region contains the carboxy terminal amino acid. In HIV-1 _JR-FL gp120, the unmodified C5 region contains the amino acids GGGDMRDNWRSELYKYKVVKIEPLGVAPTKAKRRV.
It consists of VQRE. Amino acid residues 462-500 of the sequence shown in Figure 3A have that sequence. In other HIV isolates, the C5 region will contain a homologous carboxy-terminal sequence of amino acids of similar length.

As used herein, “A492C mutation” is HIV-1 _JR-FL gp1.
It means a point mutation of 20 amino acids 492 from alanine to cysteine. HIV
The sequence is variable, so the amino acid may not be present at position 492 in all other HIV isolates. For _example, the _{HIV-1 NL4-3,} the corresponding amino acid is A499 (Genbank Accession Number AAA44992). It may be a homologous amino acid other than alanine or cysteine. The present invention encompasses cysteine mutations of such amino acids, but one of ordinary skill in the art can readily identify such amino acids in other HIV isolates.

As used herein, the “T596C mutation” refers to HIV-1 _JR-FL gp.
It means a point mutation of 41 amino acids 596 from threonine to cysteine.

Due to the variable sequence of HIV, the amino acid may not be present at position 596 in all other HIV isolates. For example, in HIV-1 _NL4-3 , the corresponding amino acid is T603 (Genbank accession number AAA4499).
2). It may be a homologous amino acid other than threonine or cysteine. The present invention encompasses cysteine mutations of such amino acids, but one of ordinary skill in the art can readily identify such amino acids in other HIV isolates.

In another aspect, a cysteine in the C1 region of gp120 is disulfide linked to a cysteine in the ectodomain of gp41.

By “C1 region” as used herein is meant the first conserved amino acid sequence present in the mature gp120 glycoprotein. The C1 region contains the amino terminal amino acid. In HIV-1 _JR-FL , the C1 region contains the amino acids VEKLWVT.
VYYGVPVWKEATTTTLFCASDAKAYDTEVHNVWATHA
CVPTDPNPQEVVLENVTEHFNMWKNNMVEQMQEDII
It consists of SLWDQSLKPCVKLTPLCVTLN. Amino acid residues 30-130 of the sequence shown in Figure 3A have that sequence. In other HIV isolates, the C1 region will contain a homologous amino terminal sequence of similar length amino acids. W4
The 4C and P600C mutations are as previously defined for the A492 and T596 mutations. Since the sequence of HIV is variable, W44 and P600
It may not be present at positions 44 and 600 in all other HIV isolates.
In other HIV isolates, homologous amino acids other than cysteine may be present instead of tryptophan and proline. The present invention encompasses cysteine mutations of such amino acids, but one of ordinary skill in the art can readily identify such amino acids in other HIV isolates.

The isolated nucleic acids include, but are not limited to, cDNA, genomic DNA, and RNA.

Those skilled in the art will know how to make nucleic acids encoding mutant viral envelope proteins in which the interaction between the viral surface protein and the transmembrane protein is stabilized. Furthermore, one of skill in the art will use these recombinant nucleic acid molecules to obtain the proteins encoded by them, as described herein for the recombinant nucleic acid molecules encoding the mutant viral envelope proteins. One will be aware of methods of doing so, and methods of carrying out treatments and prophylaxis methods using them.

The present invention provides a replicable vector containing the above nucleic acid. The present invention also provides a plasmid, cosmid, λ phage, or YAC containing the above nucleic acid molecule.
In one aspect, the plasmid is designated PPI4. The present invention is not limited to PPI4 plasmids, but may include other plasmids known to those of skill in the art.

In the present invention, a number of vector systems for expressing mutant glycoproteins may be used. For example, one class of vectors is bovine papillomavirus, polyomavirus, adenovirus, vaccinia virus, baculovirus, retrovirus (RSV, MMTV, or MoMLV), semliki forest virus, or SV.
A DNA element derived from an animal virus such as 40 virus is used. further,
Cells that have stably integrated DNA in their chromosomes may be selected by introducing one or more markers that allow for selection of transfected host cells. The marker may, for example, confer prototrophy to an auxotrophic host, confer biocide resistance (eg, antibiotics), or resistance to heavy metals such as copper. The selectable marker gene can either be directly linked to the DNA sequence to be expressed, or introduced into the same cell by cotransformation. Additional elements may also be required for optimal synthesis of mRNA. These elements can include splice signals as well as transcription promoters, enhancers, and termination signals. CDNA expression vectors incorporating such elements include (Okay
ama and Berg, Mol Cell Biol 3: 280, 1.
983).

The vectors used in the present invention are designed to express high levels of mutant viral envelope proteins in cultured eukaryotic cells and to efficiently secrete these proteins into the medium. Derivatization of the mutant envelope glycoprotein into the medium is carried out by adding an appropriate signal sequence to the mature N-terminus of the mutant envelope glycoprotein, such as the signal sequence from the tissue plasminogen activator (tPA) genomic open reading frame. It involves merging in-frame.

The mutant envelope protein is a) transfecting a mammalian cell with an expression vector for producing the mutant envelope glycoprotein; b) the transfected mammal obtained under conditions such that the mutant envelope protein is produced. Culturing the cells; c) can be obtained by recovering the mutant envelope protein thus produced.

Once the expression vector or DNA sequence containing the construct has been prepared for expression, the expression vector can be transfected or introduced into a suitable mammalian cell host. Various techniques may be used to achieve this, such as, for example, protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, or other conventional techniques. In the case of protoplast fusion, cells are grown in medium,
Screen for those with the appropriate activity. A mutant protein is produced by expressing a gene encoding a mutant envelope protein.

Methods and conditions for culturing the resulting transfected cells and recovering the mutant envelope protein thus produced are well known to those skilled in the art,
It may be varied or optimized depending on the particular expression vector and mammalian host cell to be used.

In the claimed invention, the preferred host cells for expressing the mutant envelope proteins of the invention are mammalian cell lines. Mammalian cell lines include, for example, monkey kidney CV1 strain (COS-7) transformed with SV40,
Human embryonic kidney line 293, newborn hamster kidney cells (BHK), Chinese hamster ovary cells-DHFR ⁺ (CHO), Chinese hamster ovary cells-
DHFR ⁻ (DXB11), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), cervical cancer (HELA), dog kidney cells (MDCK),
Human lung cells (W138), human liver cells (HepG2), mouse breast cancer (MMT0
60562), mouse cell line (C127), and myeloma cell line.

Other eukaryotic expression systems using non-mammalian vector / cell line combinations can be used to produce mutant envelope proteins. These include, but are not limited to, baculovirus vector / insect cell expression system and yeast shuttle vector / yeast cell expression system.

Although methods and conditions for purifying mutant envelope proteins from culture media have been described in the present invention, these procedures can be modified or optimized, as is well known to those of skill in the art. Must be recognized.

The present invention provides a host cell containing the above vector. In one aspect, the cells are eukaryotic cells. In another aspect, the cells are bacterial cells.

The present invention provides a vaccine comprising the above isolated nucleic acid. In one aspect, the vaccine comprises a therapeutically effective amount of the nucleic acid. In another aspect, the vaccine comprises a therapeutically effective amount of the protein encoded by the nucleic acid. In another aspect, the vaccine comprises a combination of a recombinant nucleic acid molecule and a mutant viral envelope protein.

Numerous adjuvants have been developed to increase the immunogenicity of protein and / or nucleic acid vaccines. As used herein, suitable adjuvants for use with protein-based vaccines include alum, Freund's incomplete adjuvant (FIA), saponin, QuilA, QS21, Rib.
i Detox, Monophosphoryl lipid A (MPL),
And non-ionic block copolymers such as L-121 (Pluronic; Syntex SAF). In a preferred embodiment,
Said adjuvant is alum, especially alum in the form of a thixotropic, viscous, homogeneous aluminum hydroxide gel. The vaccine of the present invention may be administered as an oil-in-water emulsion. Methods of mixing an adjuvant with an antigen are well known to those of skill in the art.

The adjuvant may be in particulate form. The antigen is polylactide-co-
It can be incorporated into biodegradable particles composed of glycolide (PLG) or similar polymeric materials. It is known that such biodegradable particles stimulate the long-term immune response to the immunogen by gradually releasing the immunogen. Other particulate adjuvants include Quil A, known as immunostimulatory complexes (ISCOMs),
And cholesterol, and micellar mixtures of aluminum or iron oxide, but are not limited thereto. Methods of mixing an antigen with a particulate adjuvant are well known to those of skill in the art. Formulation of protein-based immunogens and administration with appropriate adjuvants such as ISCOMs and micron-sized polymeric or metal oxide particles elicit cytotoxic T lymphocytes and other cellular immune responses It is also known to those skilled in the art.

Suitable adjuvants for the nucleic acid-based vaccines used in the present invention include Qu
il A, IL-12 provided in the form of purified protein or nucleic acid, short bacterial immunostimulatory nucleotide sequences such as CpG containing a motif, IL-2 / provided in the form of purified protein or nucleic acid Ig fusion protein, M
Oil-in-water microemulsions such as F59, polymeric microparticles, cationic liposomes, monophosphoryl lipid A (MPL), immunomodulators such as Ubenimex, and heat labile toxins of E. coli, and cholera toxin from Vibrio. It includes, but is not limited to, toxins that have been detoxified by genetic engineering. Such adjuvants and methods of mixing adjuvants with antigens are well known to those of skill in the art.

A “therapeutically effective amount” of mutant envelope protein can be determined according to methods known to those of skill in the art.

As used herein, “therapeutically effective amount” means a dose and dosing schedule sufficient to delay, halt, or reverse the progression of viral disease. In a preferred embodiment, the virus is HIV.

The present invention provides a method of treating a viral disease, comprising treating said patient by immunizing a patient infected with the virus using the above vaccine or a combination thereof.

As used herein, “treating” means either slowing, stopping, or reversing the progression of a viral disease. In a preferred embodiment, "treat"
By means to reverse the progression until the time the disease disappears. As used herein, "treating" also means reducing the number of viral infections, reducing the number of infectious viral particles, reducing the number of cells infected with the virus, or ameliorating the symptoms associated with the virus.

As used herein, “immunize” means that a patient is given a primary dose of a vaccine, followed by an appropriate period of time, followed by one or more subsequent doses of a vaccine to provide the subject with an immune response to the vaccine. It means to cause it. The appropriate period between administrations of vaccines can be readily determined by one of ordinary skill in the art and is usually weeks to months.

Depending on the nature of the vaccine and the size of the patient, the dose of vaccine may range from about 1 μg to about 10 mg. In a preferred embodiment, the dose is about 300 μg.

As used herein, “virally infected” means that the genetic information of the virus is introduced into the target cell, for example by fusing the membrane of the target cell with the virus or infected cell. To do. The target can be a cell of the patient's body. In a preferred embodiment, the target cells are bodily cells obtained from a human patient.

As used herein, “patient” means any animal or artificially modified animal that can be infected with the virus. Artificially modified animals include, but are not limited to, SCID mice with the human immune system. The animals include mice, rats, dogs, guinea pigs, ferrets, rabbits, and primates,
It is not limited to these. In a preferred aspect, the patient is a human.

[0111]   The invention provides a vaccine comprising a prophylactically effective amount of the above isolated nucleic acid.

The present invention provides a vaccine comprising a prophylactically effective amount of the protein encoded by the above isolated nucleic acid.

[0113]   A prophylactically effective amount of the vaccine can be determined according to methods well known to those skilled in the art.

As used herein, a "prophylactically effective amount" is sufficient to reduce the likelihood of infection of a patient, or to reduce the severity of an illness in a patient who is currently infected. Sufficient dose and dosing schedule.

The present invention comprises a method of reducing the likelihood of a subject being infected with a virus, comprising administering said vaccine or a combination thereof to reduce the subject's likelihood of becoming infected with the virus. Provide a method.

As used herein, “subject infected with a virus” means that the subject's own cells are invaded by the virus.

As used herein, “reducing a subject's chance of becoming infected with a virus” means
This means that the subject's chance of getting the virus is reduced by at least a factor of 2. For example, if the subject has a 1% chance of being infected with the virus, the likelihood of the subject being infected with the virus is reduced by a factor of 2 by reducing the likelihood of the subject being infected with the virus by a factor of two.
It will be 5%. In a preferred aspect of the present invention, reducing a subject's likelihood of becoming infected with a virus means reducing the subject's chance of becoming infected with the virus by at least a factor of 10.

As used herein, “administration” can be performed or carried out using any method known to those of skill in the art. The method can include intravenous, intramuscular, oral, intranasal, transdermal, or subcutaneous means.

The present invention includes the following: recombinant subunit proteins, DNA plasmids,
There is provided the above vaccine comprising, but not limited to, an RNA molecule, a replicating viral vector, a non-replicating viral vector, or a combination thereof.

The present invention is a method of reducing the severity of a viral disease in a patient, wherein the patient is exposed to the virus prior to exposure to the virus by administering the above vaccine or a combination thereof. Sometimes, a method is provided that comprises reducing the severity of a viral disease in a patient. In a preferred aspect, the virus is HIV.

As used herein, "reducing the severity of a viral disease in a patient" means slowing the progression of the viral disease and / or reducing the symptoms of the viral disease. "Reducing the severity of a viral disease in a patient" also means reducing the ability of the patient to transfer the virus to uninfected individuals.

As used herein, “exposure to a virus” means contact with a virus such that an infection can occur.

"Subsequently exposed" as used herein means exposed after one or more immunizations.

The present invention is a mutant viral envelope protein that differs from the corresponding wild-type protein by at least one amino acid and has increased stability over the corresponding complex obtained from the wild-type envelope protein. Then, a mutant virus envelope protein that provides a complex comprising a surface protein and a transmembrane protein by proteolysis is provided.

The present invention is a complex comprising a viral surface protein and a viral transmembrane protein, which has increased stability as compared to the corresponding complex obtained from the wild type envelope protein, At least one wild-type protein sequence
Provided is a complex obtained by proteolytic cleavage of a mutant viral envelope protein having different sequences of three amino acids.

The present invention provides a viral envelope protein comprising a viral surface protein and a corresponding viral transmembrane protein, wherein the viral envelope protein is a complex formed between the viral surface protein and the transmembrane protein. Provided are viral envelope proteins that contain one or more mutations that increase stability in the amino acid sequence.

The present invention is a complex comprising a viral surface protein of a viral envelope protein and a corresponding viral transmembrane protein, wherein the viral envelope protein is formed between the viral surface protein and the transmembrane protein. Complexes containing one or more mutations in the amino acid sequence that increase the stability of the complex are provided.

The present invention provides a mutant viral envelope protein encoded by the above nucleic acid molecule.

In one aspect, the mutant viral envelope protein is linked to at least one protein or protein fragment to form a fusion protein.

The present invention provides virus-like particles comprising the transmembrane protein of the present invention and a surface protein complex. In some embodiments, the virus-like particle comprises an immunodeficiency virus structural protein. In one aspect, the structural protein is a gag protein.

As used herein, “virus-like particle” or VLP (virus-like).
particles) are particles that do not contain all of the protein components of live viral particles, are non-infectious in any host, and non-replicating in any host. As used herein, VLPs of the invention, such as the HIV-1 gag required to form membrane-encapsulated virus-like particles, with a disulfide-stabilized complex of the invention. And structural proteins.

Advantages of VLPs are (1) granular and multivalent (which stimulates immunity), and (2) stabilized with a membrane-attached form of the disulfide that is close to its native state. The ability to provide an envelope glycoprotein is included.

VLPs represent viral proteins (eg HIV-1 gp1) in the same cell.
20 / gp41 and gag) are co-expressed. This is the transfection of an appropriate expression vector encoding a viral protein, V
Of several means for expressing heterologous genes well known to those of skill in the art, such as infection of cells with one or more recombinant viruses encoding LP proteins (eg, vaccinia), or transduction of cells with retroviruses. Can be achieved by either: A combination of such approaches can also be used. VLPs can be made either in vitro or in vivo.

VLPs can be made in purified form by methods well known to those of skill in the art, including centrifugation on sucrose or other layer forming material, and chromatography.

As used herein, “variant” means non-wild type. As used herein, "ligated" refers to, but is not limited to, fusion proteins formed by recombinant methods and chemical cross-linking. Suitable chemical crosslinks are well known to those skilled in the art.

In one aspect, the protein is purified by one of the methods known to those skilled in the art.

The present invention provides a vaccine comprising a therapeutically effective amount of the above protein or complex. The invention also provides a vaccine comprising a prophylactically effective amount of the above protein or complex.

The invention provides methods for stimulating or enhancing the production of antibodies that recognize the above proteins or complexes in a subject.

The present invention provides methods for stimulating or enhancing the production of cytotoxic T lymphocytes that recognize the above proteins in a subject.

The present invention provides an antibody capable of specifically binding to the above mutant protein. The present invention also provides an antibody that can specifically bind to the above mutant protein or complex, but does not bind to the wild-type protein or complex.

The present invention provides an antibody, antibody chain, or fragment thereof identified using a viral envelope protein encoded by the above recombinant nucleic acid molecule. The antibody can be of the IgM, IgA, IgE, or IgG class or subclasses thereof. The above antibody fragments include, but are not limited to, Fab, Fab ′, (Fab ′) ₂ , Fv, and single chain antibodies. The present invention provides labeled antibodies.

The present invention provides an isolated antibody light chain of the above antibody, or a fragment or oligomer thereof. The invention also provides an isolated antibody heavy chain of the above antibody, or a fragment or oligomer thereof. The invention also provides one or more CDR regions of the above antibodies. In one aspect, the antibody is derivatized. In another aspect, the antibody is a human antibody. The antibodies include, but are not limited to, monoclonal and polyclonal antibodies. In some embodiments, the antibody is humanized.

As used herein, “oligomer” means a complex of two or more subunits.

As used herein, “CDR”, or complementarity determining region, refers to a highly variable amino acid sequence in the variable domain of an antibody.

A "derivatized" antibody, as used herein, is a modified antibody. Methods of derivatization include, but are not limited to, the addition of fluorescent moieties, radionuclides, toxins, enzymes, or affinity ligands such as biotin.

As used herein, “humanized” refers to an antibody in which some, most, or all amino acids outside the CDR regions have been replaced with corresponding amino acids from a human immunoglobulin molecule. . In some embodiments of the humanized form of the antibody, some, most, or all of the amino acids outside the CDR regions are replaced with amino acids from a human immunoglobulin molecule, but within one or more CDR regions. Some, most, or all amino acids are unchanged. Minor additions, deletions, insertions, substitutions, or modifications of amino acids are permissible as long as the antibody does not lose its ability to bind an antigen. Suitable human immunoglobulin molecules include IgG1, IgG2, IgG3, IgG4, I
gA, IgE, and IgM molecules will be included. A "humanized" antibody will retain the same antigen specificity as the original antibody.

One of ordinary skill in the art would know how to make the humanized antibodies of the invention. Various publications, some of which are incorporated herein by reference,
A method for making a humanized antibody is described. For example, the method described in US Pat. No. 4,816,567 involves making chimeric antibodies having the variable region of one antibody and the constant region of another antibody.

US Pat. No. 5,225,539 describes another approach for producing humanized antibodies. The patent is directed to the use of recombinant DNA technology to produce humanized antibodies, such that the humanized antibody recognizes the desired target but is not significantly recognized by the human patient's immune system. It describes uses in which the CDRs of immunoglobulin variable regions have been replaced with CDRs of immunoglobulin variable regions having different specificities. Specifically, site-directed mutagenesis is used to implant the CDRs on the framework.

US Pat. Nos. 5,585,089 and 5,693,761 and WO90
/ 07861 describe another approach for humanizing antibodies, which describes a method for producing humanized immunoglobulins. These have one or more CDRs, a framework from the recipient human immunoglobulin, and may further have amino acids of the donor immunoglobulin. These patents describe methods of increasing the affinity of an antibody for a desired antigen. Some of the amino acids in the framework are chosen to be the same amino acids located at that site in the donor rather than the acceptor. In particular,
These patents describe the preparation of humanized antibodies that bind to the receptor by combining the CDRs of mouse monoclonal antibodies with the framework and constant regions of human immunoglobulins. The human framework regions can be chosen for maximum homology with mouse sequences. Computer models can be used to identify amino acids in the framework regions that appear to interact with the CDRs or specific antigens, followed by humanization using mouse amino acids at these positions. Antibodies can be made.

Nos. 5,585,089, 5,693,761 and WO
90/07861 also proposes four possible criteria that can be used in designing humanized antibodies. The first proposal is to use as acceptor a framework derived from a particular human immunoglobulin that is unusually homologous to the immunoglobulin of the donor to be humanized, or a consensus framework derived from many human antibodies. Was to use. The second proposal is that if the amino acids in the framework of human immunoglobulin are unusual and the donor amino acids of that part are typical human sequences, the donor amino acids may be selected instead of the acceptor. It was good. The third suggestion was that the donor amino acid may be selected over the acceptor amino acid at positions immediately adjacent to the three CDRs in the humanized immunoglobulin chain. The fourth proposal lies in a framework position where amino acids are predicted to have side chain atoms that are within 3 Angstroms of the CDRs and are capable of interacting with the CDRs in a three-dimensional model of the antibody. Was to use donor amino acid residues. The above methods are merely illustrative of some of the methods that one of skill in the art can use to make humanized antibodies.

In some embodiments of the above antibodies, the viral envelope protein is from HIV-1.

As used herein, “derived from” means obtained, in whole or in part, from HIV in the form of genomic sequences, primary isolates, molecular clones, consensus sequences, chimeras, and It includes sequences modified by such means as truncations and point mutations.

The present invention provides an isolated nucleic acid molecule encoding the above antibody. The nucleic acid molecule includes, but is not limited to, RNA, genomic DNA, and cDNA.

The present invention is a method of reducing the likelihood of viral exposure of a subject exposed to the virus, wherein said subject is infected with the virus by administering said antibody or said isolated nucleic acid. A method comprising reducing the likelihood is provided. In a preferred embodiment, the virus is HIV.

As used herein, “reducing the likelihood” means less likely than it would be under a control condition without administration of said nucleic acid, protein, or antibody. To do.

The present invention provides a method of treating a patient infected with a virus, comprising treating said patient by administering said antibody or said isolated nucleic acid. In a preferred embodiment, the virus is HIV.

The present invention provides a substance capable of binding the mutant virus envelope protein encoded by the above recombinant nucleic acid molecule. In one aspect, the substance inhibits a viral infection. In one aspect, the viral envelope protein is HI
It is derived from V-1.

As used herein, “agent” includes, but is not limited to, small organic molecules, antibodies, polypeptides, and polynucleotides.

As used herein, “inhibiting viral infection” refers to the amount of viral genetic information introduced into a target cell population relative to the amount that would otherwise be introduced. It means to reduce.

The present invention provides a method of determining whether a compound can inhibit a viral infection, comprising: a. Contacting the mutant viral envelope protein encoded by the recombinant nucleic acid of claim 1 with a suitable concentration of the compound under conditions that allow the compound to bind to the protein; b. Contacting the resulting complex with a reporter molecule under conditions that allow the reporter molecule to bind to said mutant viral envelope protein; c. Measuring the amount of bound reporter molecule; d. Comparing the amount of bound reporter molecule in step (c) with the amount measured in the absence of said compound, the reduction of the amount being an indicator that said compound may inhibit viral infection. By providing a method for determining whether a compound can inhibit a viral infection.

Using commercially available equipment, methods, and reagents (Biacore, Piscatawa
y, N.N. J. ), A method such as surface plasmon resonance may be used to measure the direct binding of said compound to the mutant viral envelope protein.

As used herein, "reporter molecule" means a molecule that can be detected when bound to a mutant envelope protein. Such molecules include radiolabeled or fluorescently labeled molecules, enzyme-linked molecules, biotinylated molecules or similarly affinity tagged molecules, or antibodies or so labeled. Molecules, which react with substances in, but are not limited to.

As used herein, “measurement” can be done by any method known to those of skill in the art. These include, but are not limited to, fluorometric, colorimetric, radiometric, or surface plasmon resonance methods.

In one aspect, the reporter molecule is an antibody or derivative thereof. In one aspect, the virus is HIV-1. In some embodiments, the reporter molecule comprises one or more host cell viral receptors or molecular mimics thereof.

As used herein, "molecular mimics" means molecules that have similar binding properties.

The present invention provides a method of determining whether a compound can inhibit a viral infection, comprising: a. Contacting the viral receptor of the host cell or a molecular mimic thereof with a suitable concentration of said compound under conditions which allow the binding of said compound with the receptor or receptor mimic; b. Contacting the resulting complex with the mutant viral envelope protein encoded by the recombinant nucleic acid of claim 1 under conditions that allow binding of the envelope protein and a receptor or receptor mimic in the absence of the compound. And c. Determining the amount of binding of the envelope protein to a receptor or receptor mimetic; d. Comparing the amount of binding determined in step (c) with the amount measured in the absence of said compound, a decrease in the amount being an indication that said compound may inhibit viral infection. This provides a method of determining if a compound can inhibit a viral infection.

In some aspects of the above methods, the virus is HIV-1. In one aspect,
The host cell viral receptor is CD4, CCR5, CXCR4, or a combination thereof, or a molecular mimetic.

As used herein, "CD4" refers to a mature, native, membrane-bound domain that contains a cytoplasmic domain, a hydrophobic transmembrane domain, and an extracellular domain that binds to the HIV-1 gp120 envelope glycoprotein. Means CD4 protein. CD
4, CD4 capable of binding to HIV-1 gp120 envelope glycoprotein
Also included is a portion of the extracellular domain.

As used herein, “CCR5” binds a member of the C—C group of chemokines, the amino acid sequence of which includes the amino acid sequence set forth in Genbank Accession No. 1705896, and related It is a polymorphic variant. As used herein, CCR5 includes the extracellular portion of CCR5 capable of binding HIV-1 envelope protein.

As used herein, “CXCR4” is a chemokine receptor that binds a member of the C—X—C group of chemokines, the amino acid sequence of which includes the amino acid sequence set forth in Genbank Accession No. 400654. And related polymorphic variants.
As used herein, CXCR4 includes the extracellular portion of CXCR4 that can bind HIV-1 envelope proteins.

[0171]   The present invention provides compounds isolated using the above method.

Pharmaceutically acceptable carriers are well known to those of skill in the art and include 0.01-0.1M, preferably 0.05M phosphate buffer, phosphate buffered saline, Or 0
． Includes, but is not limited to, 9% saline. Moreover, such pharmaceutically acceptable carriers can include, but are not limited to, aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions, suspensions, saline, and buffered solvents. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers such as those based on Ringer's dextrose, and the like. Preservatives and other additives such as, for example, antimicrobial agents, antioxidants, chelating agents, inert gases and the like may be present.

The present invention provides a compound determined to be capable of inhibiting viral infection by the above method.

The present invention provides a pharmaceutical composition comprising an amount of a compound effective to inhibit a viral infection determined to be capable of inhibiting a viral infection by the above method and a pharmaceutically acceptable carrier. I will provide a. In one aspect, the viral infection is an HIV infection. In a preferred embodiment, the viral infection is HIV-1 infection.

The present invention provides a mutant complex comprising a surface protein of an immunodeficiency virus and a transmembrane protein of an immunodeficiency virus, wherein the mutant complex of the virus is higher than the stability of the wild type complex. Mutant complexes are provided that contain in the amino acid sequence one or more mutations that increase the stability of the complex formed between the surface protein and the transmembrane protein. In one aspect, the stability of the complex is increased by introducing at least one disulfide bond between the transmembrane protein and the surface protein. In one aspect, the amino acid residue in the transmembrane protein is mutated to a cysteine residue, resulting in the formation of a disulfide bond between the transmembrane protein and the surface protein. In some embodiments, amino acid residues in the surface protein are mutated to cysteine residues, resulting in the formation of disulfide bonds between the transmembrane protein and the surface protein. In one aspect, an amino acid residue in the transmembrane protein is mutated to a cysteine residue, and an amino acid residue in the surface protein is mutated to a cysteine residue to form a disulfide group between the transmembrane protein and the surface protein. Results in the formation of bonds.

In one aspect, the immunodeficiency virus is a human immunodeficiency virus. Human immunodeficiency virus includes, but is not limited to, the JR-FL strain. The surface proteins include, but are not limited to, gp120. C1 of gp120
Amino acid residues in the region may be mutated. Amino acid residues in the C5 region of gp120 may be mutated. The amino acid residues that can be mutated include the following amino acid residues: V35, Y39, W44, G462, I482, P484, G486.
, A488, P489, A492, and E500, but are not limited thereto. The amino acid residue of gp120 is also shown in Figure 3A. Transmembrane proteins include, but are not limited to, gp41. Amino acids in the ectodomain of gp41 may be mutated. Amino acid residues that can be mutated include, but are not limited to, the following amino acid residues: D580, W587, T596, V599, and P600. The gp41 amino acid residue is also shown in Figure 3B.

The invention differs from the corresponding wild-type protein in at least one amino acid,
A mutant viral envelope protein, which has increased stability compared to the corresponding complex obtained from the wild-type envelope protein and gives a complex with a surface protein and a transmembrane protein, said surface protein and transmembrane protein The protein provides a mutant viral envelope protein encoded by different nucleic acids.

The present invention provides a mutant viral envelope protein having increased stability as compared to the corresponding complex obtained from the wild-type envelope protein and having a sequence that differs in at least one amino acid from the sequence of the corresponding wild-type protein. The present invention provides a complex comprising a viral surface protein and a viral transmembrane protein obtained by proteolysis of, wherein the surface protein and the transmembrane protein are encoded by different nucleic acids.

The present invention provides a nucleic acid encoding a mutant surface protein, the nucleic acid being complexed with the corresponding transmembrane protein and which will have increased stability. .

The present invention is a nucleic acid encoding a mutant transmembrane protein, wherein said transmembrane protein is in complex with its corresponding surface protein and will have increased stability. I will provide a.

The present invention provides an antibody that binds to the above protein or the above complex, but does not cross-react with each monomer surface protein or each monomer transmembrane protein.

[0182]   The present invention provides the above antibody capable of binding to the virus.

The present invention differs from the corresponding wild-type protein in at least one amino acid,
A protein comprising at least a portion of a viral envelope protein, which has increased stability compared to the corresponding complex obtained from the wild-type envelope protein, giving a complex with a surface protein and a transmembrane protein, , A protein in which said portion of said protein confers increased stability.

The present invention provides a portion of the above protein, wherein the portion results in increased immunogenicity as compared to the corresponding wild type portion.

The present invention further provides a simple method for determining whether a subject has produced antibodies capable of blocking viral infectivity. The diagnostic test involves examining the ability of the antibody to bind to the stabilized viral envelope protein.
As shown herein, such binding indicates the ability of the antibody to neutralize virus. In contrast, antibody binding to the unstabilized monomeric form of the viral envelope protein does not predict the ability of the antibody to bind infectious virus and block viral infectivity (Fouts et. a
l. J. Virol. 71: 2779, 1997). The method offers the practical advantage of avoiding the need to use infectious viruses.

Many immunoassay formats known to those of skill in the art are suitable for the diagnostic application. For example, a solid phase enzyme immunoassay in which mutant virus envelope glycoproteins are captured directly or biospecifically on the wells of a microtiter plate (
The ELISA format could be used. If desired, test samples are added to the plate at a range of concentrations after washing and / or blocking steps. Antibodies can be added in a variety of forms including, but not limited to, serum, plasma, and purified immunoglobulin fractions. After appropriate incubation and washing steps, bound antibody can be detected, eg, by adding an enzyme-linked reporter antibody that is specific for the subject's antibody. Suitable enzymes include horseradish peroxidase, and alkaline phosphatase, for which numerous immunoconjugates and colorimetric substrates are commercially available. The binding of the test antibody can be compared to that of known monoclonal or polyclonal antibody standards assayed in parallel. In the example, high levels of antibody binding would be indicative of high neutralizing activity.

As an example, a diagnostic test can be used to determine whether a vaccine elicits a protective antibody response in a subject, and if a protective response is present, the subject is successfully It has been shown to be immunized, suggesting the absence of such a response requires further immunization. In a preferred aspect, the subject is a human.

The present invention will be better understood by the following experimental details. However, one of ordinary skill in the art will readily appreciate that the specific methods and results discussed are merely illustrative of the invention more fully described in the claims which follow. You can understand.

[0189]

[Experiment details]

<Materials and methods> 1. Material The plasmid designated pp14-tpa-gp120 _JR-FL is a microorganism American Type Culture Collection ((ATCC), 12301 Parklawn Drive, Rockville, Maryland
20852) and was deposited as ATCC authorization number 75431. This plasmid, 1993
Deposited with ATCC on March 12th. The eukaryotic shuttle vector contains the major immediate early (CMV MIE) promoter / enhancer of cytomegalovirus linked to a full-length HIV-1 envelope gene that replaces the signal sequence with the signal sequence of tissue plasminogen activator. There is. In this vector, a stop codon was placed at the C-terminus of gp120 present in this vector to prevent translation of the gp41 sequence. This vector contains ampicillin resistance gene, SV40 origin of replication and β
-Includes DHFR driven by the globulin promoter.

Epitopes and various immunochemical properties of anti-gp120 monoclonal antibodies obtained from various donors have been previously described. (Moore et al., J.
Virol. 768: 469, 1994; Moore and Sodroski, J. et al. Virol. 70: 1863, 1996). These include the following monoclonal antibodies: V3 locus (Moore et al., J. Vir
Mab 19b for ol.69: 122, 1995); Mabs 50.1 and 83.1 for the V3 loop (Wh
ite-Scharf et al. Virology192: 197, 1993); Ma for the CD4 binding site (CD4b)
bs IgG1b12 and F91 (Burton et al., Science 266: 124, 1994; Moore and Sodros
ki, J. 70: 1863, 1996); Mab 2G12 against a unique C3-V4 glycan-dependent epitope (Trkola et al., J. Virol, 70: 1100, 1996); Mab M90 against the C1 region.
(DiMarzo Veronese et al. AIDS Res. Human Retrov, 8: 1125, 1992); MAb 23a and Ab D7324 for the C5 region (Moore and Sodroski, J. Virol. 70: 1863,
1996); Mab 212A (Moore et al. J. Virol 68) to the structural C1-C5 epitope.
: 6836,1994); Mab 17b (Moore and Sodrosk for CD4-induced epitopes)
i, J. Virol. 70: 1863, 1996); Mab A to the CD4-induced C1-C4 epitope
32 (Moore and Sodroski, J. Virol. 70: 1863, 1996; Sullivan et al, J. Viro.
72: 4694, 1996); Mabs antibody G3-299 (Mo against C4 or C4 / V3 epitope)
Ore and Sodroski, J. Virol. 70: 1863, 1996); Mabs for the gp41 epitope containing 7B2 for epitope cluster 1 (provided by Jim Robinson of Tulane University); 25C2 for the fusion peptide region (Buchacher e.
t al. AIDS Res. Human Retrov., 10: 359, 1994); 2P5 against neutralizing epitopes surrounding 665-690 residues (Munster et al J. Virol. 68: 4031, 1994). Tetrameric CD
4-IgG2 has been detailed previously.

Anti-HIV antibodies were obtained from commercial sources, the NIH AIDS reagent program, or the inventor. When so indicated, antibodies were biotinylated with NIH-biotin according to the manufacturer's manual (Pierce, Rockford, IL).

The Gp120 _JR-PL monomer was produced in CHO cells that stably expressed the PP14-tPA-gp120 _JR-PL plasmid as detailed previously (US Patents 5,866,163 and 5,86).
9,624). Soluble CD4 was purchased from Bartels Corp (Issaquah WA).

2. Construction of PP14-based plasmids expressing wild-type and mutant HIV envelope (jacket) proteins Wild-type gp140s (gp140WT) The sequence coding for gp140 is JR-PL, DH123, GUN-1, 89.6, NL4-3 Was amplified using the polymerase chain reaction (PCR) from full-length HIV-1 molecular clones such as HXB2 and HXB2. The 5'-primer was named Kpnlenv (5'-GTCTATTATGGGGTACCTGTGTGG
AAACAAGC-3 '). On the other hand, the 3'-primer is BstBlenv (5'-CGCAGACGCAGATTCGAATTA
ATACCACAGCCAGTT-3 '). PCR was performed under stringent conditions to remove the error introduced by Tag polymerase. The PCR product was cleaved with the restriction enzymes Kpnl and Xhol and purified by agarose electrophoresis. The plasmid PP14-tPA-gp120JR-PL was also digested with these two restriction enzymes and the larger fragment (vector) was gel purified as well. PP14-tPA-gp120
The JR-PL expression vector has been previously detailed (Hasel and Maddon, US Patents # 58.
86163 and 5869624). Ligation of the insert with the vector was performed overnight at room temperature. DH5αF'Q10 bacteria were transformed with a 1/20 chance of each mating. Colonies were screened directly by PCR to see if they were transformed by the vector containing the insert. DNA from three positive clones of the nuclear construct was purified using a plasmid preparation kit (Qiagen, Valencia, CA), and both full-length strands of gp160 were sequenced. For example, pPP
14-gp140WT _JR-PL and pPP14-gp140WT _DH123 are related to vectors expressing wild-type cleavable gp140 derived from HIV-1 _JR-PL and HIV-1 _DH123 , respectively.

The truncation mutants of gp140UNC gp120-gp41, JR-FL gp140 have been previously described in detail (Ea
rl et al., Proc. Natl, Acad. Sci USA 87; 648, 1990), made by substitution within the C-terminal REKR motif of gp120. The deletion is the mutagenesis of a specific site, and the mutagenic primer 5'140M (5'-CTACGACTTCGTCTCCGCCTTCGACTACGGGAATAGGAGCTGTGT
TCCTTGGGTTCTTG-3 ') and 3'gp140M (Kpnlenv and BstBlenv 5'-TCGAAGGCGGAGACGAAGTCGTAGCCGCAGTGCCTTGGTGGGTGCTACTCCTAATGGTTC-3'). In the ligation with Kpnlenv and BstBlenv, the PCR product was cleaved with Kpnlenv and BstBlenv and pPP14
Subcloned into.

Loop-deficient gp120 and gp140 Variability PPI4-based plasmids expressing the loop-defective gp120 and gp140 proteins were prepared using splicing by the Overlap-Extension method, which has already been detailed (Binley et al. ., AIDS Res, Human Retrovir. 14: 191, 1998).
In the single loop-deficient mutant, the Gly-Ala-Gly spacer is D132-K152 (ΔV1), F15.
Used to replace 6-I191 (ΔV2), or T300-G320 (ΔV3). This numbering corresponds to that of the JR-FL clone or HIV-1 (Genbank accession number U63
632).

DGKPN5 ′ and 5JV1V2-B (5′-GTCTATTATGGGGTACCTGTGGAAAGAA on ΔV1 template)
PCR amplification with GC-3 ') followed by cleavage with Kpnl or BamH1 produced a 292 bp fragment lacking the V1 loop coding sequence. This fragment was plasmid cloned into the sequence for the V2 loop using the Kpnl and BamH1 restriction sites. The constructed plasmid was named ΔV1V2 ′, which is D132-K152.
And contains the G1y-A1a-GIy sequence instead of F156-I191. Envs lacking the V1, V2, and V3 loops were also detected by PCR on the ΔV3 template using the primers 3JV2-B (5'-G
TCTGAGTCGGATCCTGTGACACCTCAGTCATTACACAG-3 ') and H6NEW (5'-GTCTGAGTCTTC
GAATTAGTGATGGGTGATGGTGATGATACCACAGCCATTTTGTTATGTC-3 '). This fragment was cloned into ΔV1V2 ′ using BamH1 and BatB1. The generated env construction was named ΔV1V2′V3. The glycoprotein encoded by the ΔV1V2 ′ and ΔV1V2′V3 plasmids encodes a short sequence of amino acids spanning C125 and C130. These sequences were removed using T127-I191, G1y-A1a-G1y, mutagenesis primers substituting the sequences. We use the primer 3'DV1V2STU1 (5
'-GGCTCAAAGGATATCTTTGGACAGGCCTGTGTAATGACTGAGGTGTCACATCCTGCACCACAGAGTGGG
PCR amplification with GTTAATTTTACATGGC-3 ') and DGKPN5'PP14 was performed, the resulting fragment was cut with Stu1 and Kpn1 and cloned into the PP14 gp140 vector. occured
gp140 was named ΔV1V2 *. ΔV1V2 * V3 was constructed in a similar manner. Amino acid substitution sites are shown schematically in FIG.

Glycosylation Site Variants Standard N-linked glycosylation sites are 357 and 398.
Was removed by introducing a point mutation from asparagine to glutamine. These conversions were done on a template that encodes both wild-type and loop-defective HIV envelope proteins.

Designated amino acids in disulfide-stabilized gp140 gp120 and gp41 were mutated to cysteine pairs by site-directed mutagenesis using the Quickchange kit (Stratagene, La jolla, CA). As shown below, some amino acids near the introduced cysteine were mutated to alanine to better adapt the cysteine mutation using a similar method within the local topology of the envelope glycoprotein. It was These conversions were done on a template that encodes both wild-type and loop-defective HIV envelope proteins.

3. Expression of gp140 in transiently transfected 293T cells: HIV envelope protein contains adherent 293T cells, the SV40 origin.
It was transiently expressed in a human embryonic kidney cell line (ATCC cat. # CRL-1573) transfected with the SV40 large T antigen which promotes high replication levels of plasmids like PP14. 293 cells are Dulbecco's minimal essential medium (DM) containing 10% fetal bovine serum supplemented with L-glutamine, penicillin and streptomycin.
EM; Life yechnologies, Gaitensburg, MD). Cells were seeded in 10 cm dishes and transfected with 10 mg of purified PP14 plasmid using the calcium phosphate precipitation method. The next day, the cells were fed with 0.2% bovine serum albumin and fresh DMEM containing L-glutamine, penicillin and streptomycin. For radioimmunoprecipitation assays, the culture also contained ³⁵ S-labeled cysteine and methionine. In one experiment, cells were cotransfected with 10 mg of the human furin gene pcDNA3.1 expression vector (Invitrogen, Carlsbad, CA).

4. ELISA analysis The concentration of gp120 and gp140 in 239 T cell supernatant was measured by ELISA (Binley et al., J. Viol 71: 2799, 1997). Briefly, Immulon II ELISA plates (Dynatech Laboratories, Inc) were plated for gp120 for 16-20 hours at 4 ° C.
It was coated with a polyclonal sheep antibody that recognizes the C-terminal sequence (APTKAKRRVVQREKR) and blocked with 2% nonfat milk in TBS. 10 cell supernatants (100 ml)
Add a range of dilutions in Tris buffer containing% fetal bovine serum. The plate was incubated at an appropriate temperature for 1 hour and washed with TBS. Anti-gp120 or anti-gp41
Antibodies were added for another hour, plates were washed with TBS and the amount of bound antibody was measured using anti-human IgG or anti-mouse IgG-conjugated alkaline phosphatase. Alternatively, biotinylated reporter antibody was used in a similar manner and measured with streptavidin-AP complex. In both cases, AP activity is AM
It measured using PAK Kit (DAKO) according to a company's instruction. To examine the reactivity of denatured HIV envelope proteins, cell supernatants were boiled for 5 minutes in the presence of 1% detergent, sodium dodecyl sulfate and NP-40 before being placed in ELISA plates at constant dilutions. Let Purified recombinant JR-FL gp120 was used as a reference standard.

5. Radioimmunoprecipitation Assay (RIPA) ³⁵ S-labeled 239 T cell supernatant was collected 2 days after transfection for RIPA analysis. From the culture supernatant, the final concentration was 50 mM Tris-HCl, 5 mM ED
Cell debris was removed by low speed centrifugation before addition to TA, pH 7.2 RIPA solution. A biotinylated antibody (about 10 mg) was added to 1 ml of the supernatant and incubated at an appropriate temperature for 10 minutes. The sample was then incubated at 4 ° C for 12-18 hours with gentle agitation with streptavidin agarose beads. Alternatively,
Unlabeled antibody was used in combination with protein G-agarose (Pierce, Rockford, IL). Bound protein was 0.05 M Tris-HCl, 10% glycerol, 2%
Eluted with SDS-PAGE sample solution containing SDS, 0.001% bromophenol blue, then 100 mM dithiothreitol (DTT) where indicated, heating at 100 ° C for 5 minutes. The sample was placed on an 8% polyacrylamide gel and electrophoresed at 200 V for 1 hour. After drying the gel, it was exposed to a Phosphor screen and the
Image analysis was performed using m Phosphoimager (Molecular Dynamics, Sunnyvale, CA). ¹⁴ C labeled protein was used as a standard for size calculation.

<Experimental Results> 1. Processing of gp140NON is promoted by co-expression of furin protease To minimize the production of gp140NON, pcDNA3.1-furin and pPP14-gp140WT
JR-FL was co-transfected into 293T cells and RIPA was used with anti-gp120 Mab 2G12
The assay was run. As shown in FIG. 2, furin eliminated the production of gp140NON, but had no effect on gp140UNC.

Similar results were obtained with the RIPA assay performed with the other anti-gp120 Mabs (data not shown).

Treatment of the samples with DTT prior to SDS-PAGE did not affect the mobility or relative amount of these bands, rather than gp140 being a separate disulfide linked gp120-gp41 molecule. Indicates that it is composed of a single-chain polypeptide.

2. Stabilization of gp120-gp41 binding by the introduction of two cysteine mutations By cotransfection of furin we then
The 41 ECTO component was able to express soluble gp140 protein bound only non-covalently. It resembles what happens to the native trimeric envelope glycoprotein complex present in virions. However, gp120-gp41 binding is weak on the surface of the virus or its infected cells, and therefore gp12
0 is gradually shielded (Mckeating et al., J. Virol 65: 852, 1991). We have found that this also occurs with the gp140WT protein, which is made in the presence of endogenous furin. Thus, we were able to detect the stable gp-20-gp41 ECTO complex present in very small amounts in the supernatant of cells expressing gp140WT after immunoprecipitation. Therefore, a method for stabilizing the gp120-gp41 noncovalent bond was searched for by introducing an intermolecular disulfide bond between the gp120 and gp41 subunits.

Therefore, we focused on the amino acids previously found to be important in gp120-gp41 binding and replaced one of several different positions in the C1 and C5 regions of gp120 with a cysteine residue ( Figure 3a). At the same time, a second cysteine mutation was introduced into several residues near the intramolecular disulfide loop of GP41 (Fig. 3B). The intent is to identify pairs of cysteine residues in native GP120-GP41 whose physical proximity is naturally formed as an intermolecular disulfide bond. As a result, over 50 different double cysteine substitutions were made in the JR-FL gp140WT protein and co-expressed with furin in transient transfections of 293T cells.

Initial analysis of transfection supernatants by antigen capture ELISA revealed that g
All mutants were shown to be effectively expressed as secreted proteins except those containing a cysteine at residue 486 of p120 (data not shown)
. Next, we characterized it by immunoprecipitating the transfection supernatant with anti-gp120 Mabs (FIG. 4). Expected 120 kDa band (gp1
In addition to 20), a second band of approximately 140 kDa was precipitated by F81 and 2G12 from supernatants transfected with many double cysteine mutants. The gp140 band derived from a mutant in which cysteine is present in the C1 region of gp120
The 120 cysteines are slightly more slowly migrating and more diffuse than their counterparts in the C5 region (Fig. 4). The existence of diffusive bands with low mobility on SDS-PAGE was previously reported (Earlra, Proc. Natl. Acad. Sci.
USA 87: 648, 1990; Earl et al., J. Virol. 68: 3015, 1994).
Probably incomplete processing of the envelope glycoprotein. 140
The relative strength of the kDa band is strongly dependent on the position of the introduced cysteine, suggesting that a certain spatial requirement is required for the formation of stable intersubunit disulfide bonds. ing.

To determine which of the double cysteine mutants would be suitable for further analysis, we immunoprecipitated each mutant with a potent neutralizing anti-gp120 Mab 2G12 followed by SDS-. PAGE and densitometry were performed to determine the relative intensities of the gp140 and gp120 bands (Figure 5). We searched for the mutants with the highest gp140 / gp120 ratio, which we interpret as an indicator of optimal formation of intramolecular disulfide bonds. From FIG. 5, it is clear that the mutant A492C / T596C has this property. From now on, we will call this protein the SOS gp140 mutant. Of note, the mobility of SOS gp140 on SDS-PAGE is consistent with that of the gp140 NON protein with gp120 and gp-40 ECTO residues linked by peptide bonds. The gp140 band obtained from the SOS mutant is not as sharp as the band of the gp140 NON protein, but less diffuse than the gp140 band obtained from any other double cysteine mutant (FIG. 4). This suggests that the SOS mutant was effectively processed. The complete nucleic acid and amino acid sequence of JR-FL SOS gp140 is provided in FIG.

We demonstrated that treatment of the immunoprecipitated protein with DTT prior to gel electrophoresis stabilizes the 140 kDa protein with intramolecular disulfide bonds. In contrast, the 140 kDa bands of gp140WT and gp140UNC were unaffected by DTT treatment, as would be expected for an uninterrupted probe protein. Here, it is noted that the 149 kDa band was never seen with the single mutation of A482C or T596C (Fig. 6b). This means that the double cysteine mutant
Further proof that the 140 kDa band results from the formation of intermolecular disulfide bonds between gp120 and gp41 ECTO. 140 kD in the absence of exogenous furin
The a protein band was not reduced by DTT, which means that this band
It is suggested that it is a double cysteine mutant of gp140NON (Fig. 6c).

3. An approach to improve the efficiency of disulfide bond formation in SOS gp140 protein. Disulfide export stabilized gp140 is the only e present in 293T cell supernatant.
Not nv species. There is also an appreciable amount of free gp120. This is gp120 and gp
It means that the disulfide bond between 41 is formed with incomplete efficiency. Free type
Although gp120 can be removed by the purification method described below, it was attempted to further reduce or eliminate its production. For this purpose, additional amino acid substitutions were made near the inserted cysteine. In addition, the position of the cysteine of gp120 was changed. We retained the cysteine of gp41 at residue 596 because this position appeared to be the most favorable for intramolecular disulfide bonds.

We first modified the position of the cysteine substitution of gp120 by placing it at the N-terminal or C-terminal alanine-492. The Gp140 / gp140 + gp120 ratio was not increased in any of these mutants, which remained somewhat comparable to that in the SOS gp140 protein (FIG. 7). Next we have alanine-492
We examined whether the large side chains of the lysine group adjacent to the ss influence disulfide bond formation. There, the lysines at 491 and 493 were mutated to alanine in contrast to SOS gp140, these mutations also showed a Gp140 / gp140 + gp120 ratio of gp.
It also did not affect the mobility of 140 (Fig. 7). Finally, a second cysteine pair was introduced into SOS gp140 at residue 44 of gp120 and residue 600 of gp41.
This is because when this cysteine pair is introduced into the wild-type protein, disulfide bonds are formed quite effectively (Fig. 5). However, this quadruple cysteine mutant (W44C / A492C / P600C / T596C) had poor expression efficiency, implying a problem in protein processing or folding (Fig. 7). This poor expression efficiency was also observed in other quadruple cysteine mutants (W44C / K491C / P600C / T596C) and (W44C / K493C / P600C / T596C).

Additional approaches to optimize the efficiency or overall expression of disulfide-stabilized variants are possible. For example, stable furin-expressing cells can be prepared so that all cells expressing the SOS gp140 protein have sufficient furin. Similarly, the furin and gp140 proteins are co-expressed in a single plasmid. K491 and K493, alone or in pairs, can be mutated to residues other than alanine. In order to better adapt the introduced cysteine, gp120 or gp
Other amino acids located near the introduced cysteine of 41 can be mutated as well.

4. Antigenicity of SOS gp140 protein is similar to virus-associated gp120-gp41. Compared to Gp140NON, SDSgp140 protein has some differences as an antigen, for which we We believe it would be advantageous for proteins intended to mimic the structure of the related gp120-gp41 complex. It is summarized below.

1) SOS gp140 protein is a potent neutralizing antibody, which is a monoclonal antibody IgG1b12
And 2G12 and also strongly binds to CD4-IgG molecules (Fig. 8a). Although the RIPA method is not quantitative enough to allow accurate determination of relative affinities, the reactivity of these monoclonal antibodies with the SOS gp140 protein of CD4-IgG2 is greater than that with the gp140NON and gp120 proteins. Quite large (Figure 8a). The SOS gp149 protein clearly has a direct CD-4 binding site. The V3 loop epitope is also
It bound the SOS gp140 protein as shown by its reactivity with monoclonal antibodies 19b and 83.1 (Fig. 8a).

2) In contrast, some non-neutralizing anti-gp120 monoclonal antibodies bind little or no SOS gp140 protein but strongly react with gp140NON and gp120 (FIG. 8b). These monoclonal antibodies are present in gp120
Includes those that respond to the C1 and C5 regions that are thought to be shielded for proper formation of the 120-gp41 complex (Moore et al., J. Virol. 69: 469, 1994; W.
yatt et al., J. Virol 71: 9722, 1997). On the contrary, all the monoclonal antibodies against C1 and C5 reacted strongly with the gp140NON protein (Fig. 8b).

3) Epitope exposure to monoclonal antibody 17b by binding to soluble CD4 occurs more efficiently in SOS gp140 than in gp140NON or gp120 proteins (FIG. 8c). Indeed, in the absence of soluble CD4, 17b had very little reactivity with the SOS gp140 protein. The CD4 induced monoclonal antibody 17 epitope is a coreceptor of gp120.
) Overlaps the binding site. It is believed that this site is exposed to the virus-associated gp120-gp41 complex by a conformational change that initiates virus-cell fusion after CD4 binding. Induction of the 17b epitope was induced by SOSgp140
It is suggested that the gp120 portion of the protein has a static structure and a degree of structural change similar to those of gp120-gp41 in the virus. The gp140 NON protein binds constitutively to 17b. Some induction of the 17 bepitope is also seen upon binding of soluble CD4, but to a lesser extent than SOS gp140.

4) Another CD4-induced epitope on gp120 is the epitope recognized by monoclonal antibody A32 (Moore et al., J. Virol. 70: 1863, 1996;
Sullivan et al., J. Virol. 72: 4694, 1998). In the absence of soluble CD4, binding of A32 to the SOS gp140 mutant was negligible, but the epitope was strongly induced upon binding to soluble CD4 (Fig. 8c). As observed in 17b,
The A32 epitope was induced less efficiently on gp140NON than on SOS gp140.

5) In any set of non-neutralizing gp41 monoclonal antibodies, SOS
There was no reactivity with the gp140 protein. On the other hand, all these monoclonal antibodies bound strongly to the gp140NON protein. These anti-gp41 monoclonal antibodies recognize several ectodomains of gp41, but all of these sites appear to be masked by gp120 in the virus-associated gp120-gp41 complex (Moore et al., J. Virol. 68: 469, 1995; Sattentau et al., Virology 2
06: 713, 1995). The inability of these to bind to the SOS gp140 protein is another strong indication that this protein has a conformation similar to that of the natural trimer. Strong recognition is
This is consistent with the abnormal structure of these proteins due to the peptide bond linking gp120 and gp41 (Edinger et al., J. Vitol. 73: 4062. 1999) (Fig. 8d).

6) In contrast to what was observed with non-neutralizing monoclonal antibodies, anti-gp
41 monoclonal antibody 2F5 efficiently binds to the SOS gp140 protein, but gp1
Does not combine with 40NON. Of note, the 2F5 epitope is the natural gp in gp41.
It is the only site thought to be well exposed in the 120-gp41 complex (Sattentau et al., Virology 206: 713, 1995). 2F of it
The ability to bind 5 is consistent with the fact that the SOS gp140 protein adopts a conformation very similar to that of the native trimer.

The antigenicity of the SOS gp140 protein was compared to that of the W44C / T596C gp140 mutant. Of the mutants containing a cysteine substitution in the C1 region, this is the most efficient in gp140 formation. W44C / T596C gp140 reacts with the 2G12 monoclonal antibody but only relatively weakly with CD4-IgG2 and IgG1b12. Furthermore, the induction of the 17b epitope on W44C / T596C gp140 by soluble CD4 was insignificant and there was strong reactivity with the non-neutralizing anti-gp41 monoclonal antibody. (FIG. 8). Therefore, we determined that this variant had suboptimal antigenic activity. Indeed, a comparison of W44C / T596C gp140 and SOS gp140 proteins shows that the position of the intramolecular disulfide bond has a significant effect on the antigenic structure of the gp140 molecule.

Compared to the antigenic nature of the gp140 SOS protein, gp140WT and gp140
The 140 kDa protein of UNC is a non-neutralizing anti-gp120 and G3-519, and 7
It reacted strongly with an anti-gp41 monoclonal antibody such as B2. In addition, the epitope recognized by monoclonal antibody 17B was constitutively exposed rather than CD4-induced.

Overall, there was a strong correlation between the binding of monoclonal antibodies to the SOS gp140 protein and their ability to neutralize HIV-1 _JR-FL . This correlation is gp140NON, g
It was not observed with p140 UNC or p120 proteins.

5. Formation of disulfide bonds between subunits is not isolated virus-dependent In order to verify the generality of our observations regarding the gp140 protein derived from RS HIV-1 isolate JR-FL, we used other HIV-1 isolates. A double cysteine mutant in gp140 from a species was created. These are R5 X4 virus DH123 and X4 virus H
Includes xB2. In each case, cysteine is the alanine-49 of JR-FL.
2 and threonine-596 were introduced at the corresponding residue positions. The resulting SOS protein was transiently expressed in 293T cells and RIPA analysis confirmed its aggregation, processing and antigenicity. Figure 9
As shown in, a sufficient amount of 140 kDa material was produced in DH123 and HxB2SOS,
We have shown that our method can successfully stabilize the envelope proteins of many types of viruses.

[0224] 6. HIV envelope modified in variable loop and glycosylation region
We present the disulfide-stabilized form of the protein as there is evidence to suggest that mutations of specific variable loops and glycosylation sites provide a more convenient means of exposing conserved neutralizing epitopes. Aggregation and antigenicity were examined. In the first study, A492C / T
A 596C JR-FL gp140 mutant was generated for the ΔV1, ΔV2, ΔV3, ΔV1V2 ^* and ΔV1V2 ^* V3 molecules, respectively, described above. For the ΔV1V2 ^* V3 proteins, glycosylation site mutants were created by NQ point mutations at amino acids 357 and 398.

In each of the mutants, either alone or doubly loop-deficient, we have found that gp
The 140 band was detected in an amount comparable to the full-length SOS gp140 protein (FIG. 11B).
To investigate whether the loss of the variable loop alters its antigenicity in the oligomerized state, the ΔV3 and ΔV1V2 ^* SOS proteins were precipitated with a paneled monoclonal antibody (FIG. 12). Monoclonal antibodies against gp41, with the exception of 2F5, showed no binding to the loop-deficient type of cysteine-stabilizing protein, indicating that these epitopes are still masked. Monoclonal antibodies against C1 and C5 epitopes also did not react. Neutralizing antibody 2F5 bound to the mutant and specifically reacted to the ΔV3SOS protein. Monoclonal antibodies against CD4BS (IgG1b2, F91) along with 2G12
It also bound well to these mutants as well. Of note is CD4-IgG2 and 2G
12 bound to the multimeric ΔV3SOS protein with very high affinity. Furthermore, in agreement with data showing that the CD4i epitope is constitutively exposed to the ΔV1V2 ^* protein, binding of monoclonal antibodies 17b and A32 to the ΔV1V2 ^* SOS variant was not induced by SCD4. . However, the ΔV3SOS mutant bound weakly to 17b and A32 in the absence of SCD4 and strongly in the presence of SCD4. These results are in agreement with previous observations that the V1 / V2 and V3 loop structures are involved in masking the CD4I epitope (Wyatt et al., J. Virol. 69: 5723,
1995). Taken together, these results show that the g
It was shown that p140 is disulfide-stabilized without impairing the rigid three-dimensional structure. 14 and 15 show ΔV1V2 ^* SOS and ΔV3JR-FL SO, respectively.
Contains the amino acid sequence of the S protein.

[0226] △ V1V2 ^* V3 and △ V1V2 ^* V3 N357Q, for the N398Q SOS mutant, gp1
40 (110 kDa and 105 kDa) was not precipitated with any neutralizing or non-neutralizing antibody (Fig. 11A, 3, 4, 7, 8 lanes). However,
We detected 90 kDa and 85 kDa bands of intensity corresponding to the mutant gp120 domain. These preliminary experiments suggest a number of approaches to disulfide-stabilized, triple-loop-deficient gp140, including altering the position of one or more introduced cysteines, and another cysteine. Adding a pair; mutating an amino acid existing in the vicinity of the introduced cysteine; changing the way of loop deletion. Another possibility is that the triple loop-defective gp140s derived from isolated HIV of other species
It may be more easily stabilized by a cysteine introduced at a residue similar to 496/592.

[0227] 7. Production and Purification of Recombinant HIV-1 Envelope Protein Milligram quantities of high quality HIV-1 envelope glycoprotein were produced in CHO cells stably expressing the PPI4 envelope expression plasmid (US Pat. No. 5,886,163).
And No. 5,869,624). The PPI4 expression vector contains the dhfr gene under the control of the β-globulin promoter. Selection of dhfr clones was performed in nucleoside-free medium by gene amplification using a stepwise increase in methotrexate concentration. The cytomegalovirus (CMV) promoter drives high levels of heterologous genes and the tissue plasminogen activator signal sequence ensures efficient protein secretion. High levels of gp120 expression and secretion were obtained by simply containing the entire 5'noncoding sequence of the CMV MIE gene and the initiation ATG codon. To make milligram quantities of protein, recombinant CHO cells were seeded in roller tubes in selective media and grown to confluent. At the production stage of the target protein, a medium containing low concentration serum was used, and the supernatant was collected twice a week. Purification processes including lectin affinity, ion exchange, and gel filtration chromatography were performed under non-denaturing conditions.

8. Protocol for Determining the Antigenicity of Stabilized HIV-1 Envelope Subunit Protein Purified recombinant HIV-1 envelope protein was formulated with a suitable adjuvant (eg Alum or Ribi Detox). For Alum, the mutation HIV-
Formulated formulations were obtained by mixing 1 envelope glycoprotein (phosphate buffer, normal salt or similar carrier) with aluminum aluminum hydroxide gel at a final concentration of about 500 mg / ml (Pierce, Rockford, IL). Antigen was adsorbed to alum gel for 2 hours at room temperature.

Guinea pigs and other animals were immunized five times at monthly intervals with approximately 100 μg of the formulated antigen by subcutaneous intramuscular or intraperitoneal injection. The sera of immunized animals were collected at intervals of 2 weeks, and the reactivity of HIV-1 envelope protein was determined by the above ELI.
Neutralizing activity was also examined in SA and well established HIV-1 infection assays (Trk
ola et al., J. Virol 72: 1876, 1998). Vaccine candidates with the highest levels of HIV-1 neutralizing activity can be tested for immunogenicity and prophylactic or therapeutic efficacy against SHIV monkeys or other models of HIV infection in non-human primates, as described below. . The subunit vaccine could be used alone or in combination with other vaccine components designed to elicit a prophylactic cellular immune response.

For these studies, the HIV-1 envelope protein is administered alone and in combination with one or more cellular HIV receptors such as CD4, CCR5 and CXCR4. As mentioned above, binding of soluble CD4 exposes a previously conserved, conserved neutralizing epitope on the HIV-1 envelope protein that was stabilized. Antibodies directed against these, or other neoepitope, can have significant antiviral activity. As described above, the CD-env complex and fusion coreceptors such as CCR5 and CXCR4 (
Interaction with coreceptors is thought to cause additional structural changes in env required for HIV fusion. The trivalent complex of stabilized env, CD4 and co-receptor thus adopts an additional intermediate fusion conformation, some of which remain active for a sufficient period of time through treatment and possible immune interference. (Kilby et al., Nat. Med. 4: 1302, 1998). env-CD4 and env-CD
Methods of preparing and administering 4-coreceptor complexes are known in the art (LaCasse et al., Science 283: 357, 1999; Kang et al., J. Virol., 68: 5854, 1994).
Gershoni et al., FASEB J. 7: 1185, 1993).

9. Protocols PCR techniques to determine the immunogenicity of the nucleic acid-based vaccines encoding stabilizing HIV-1 envelope protein, pVAXl (Cat # V260-20) a nucleic acid, such as for subcloning a DNA vaccine plasmid vectors Used to. PVAX1 is 1995
FDA document "Points to Consider on Plasmid DNA Vac" published on December 22, 2010
It was created based on a specialized method found in "cines for Preventive Infectious Disease Indications". PVAX1 has the following characteristics: Eukaryotic DNA sequences are designed to minimize the possibility of chromosomal uptake.
Limited to the sequences required for expression. Ampicillin was reported to cause an allergic reaction in some people, so we used kanamycin for E. coli vector selection. The expression level of recombinant protein from PVAX1 was
Similar to that of DNA3.1, the small size of pVAX1 and a variety of unique cloning sites amplifies subcloning of even very large DNA fragments.

Several methods were used to optimize the expression of disulfide-stabilized proteins in vivo. For example, standard PCR cloning techniques could be used to insert certain elements of pVAXC1 of the optimized PPI4 expression vector containing intron A and its adjacent CMV promoter region. In addition to that, H
The genomic DNA sequence of the IV envelope was biased towards the suboptimal codon in mammals (Haas et al., Current Biol. 6: 315, 1996). These can be converted to more convenient codons using standard mutagenesis techniques to improve nucleic acid-based HIV vaccines (Andre et al., J. Virol. 72: 1479, 1
998). Codon optimized strategies can increase the number of CpG motifs known to increase the immunogenicity of DNA vaccines (Klinman et al., J. Immunol. 518: 3
635, 1997). Finally, for the transient transfection system described above, gp1
Processing of env to 20-gp41 is facilitated by heterologous expression of furin introduced into the same or different expression vectors.

Insert-containing plasmids are administered to animals using direct injection or genetic shotgun technology and the like. This technique is known to those skilled in the art.

In one protocol, red-haired monkeys were each dosed with a 1 mg dose of nucleic acid five times. This dose was given at 4 week intervals. Each dose was administered intramuscularly. After 4 months, the animals were treated with 2 mg of nucleic acid (300 μg
, With or without mutated HIV-1 envelope glycoproteins). This series is subsequently boosted with one or more recombinant protein subunits. Animals were bled at 2-4 week intervals. Serum samples were prepared for each blood draw and examined for specific antibody production, as detailed in the following section.

SHIV Challenge Experiments Several chimeric HIV-SIV viruses have already been generated and their infectivity in red-haired monkeys has been characterized. In the virus challenge experiment, red-haired monkeys
A pre-calculated amount of virus was administered intravenously to infect more than 9/10 animals. SHIV infection was determined in two assays. Detection of the antigen SIV p27 in monkey serum by ELISA was done using a commercially available kit (Coulter).
Western blot detection of anti-gag antibodies was also performed using a commercial kit (Cambridge Biotech).

The infection rate of virally immunized control monkeys or the reduction of p27 antigen produced was
Demonstrate that the vaccine or vaccine combination has prophylactic value.

[Brief description of drawings]

FIG. 1: Various forms of HIV-1 envelope glycoproteins: Illustrations are: i) monomeric gp120; ii) full length recombinant gp160.
Iii) gp140 (gp140UNC or gp140) in which a peptide bond between gp120 and gp41 is retained and which is not processed by proteolysis.
NON); iv) SOSgp140 (proteolytically processed gp140 stabilized by intermolecular disulfide bonds); v) native gp120-gp41 trimer associated with virions. gp140UN
The shaded portion of C protein (iii) is poorly exposed or not exposed at all on the SOS gp140 protein, or on the native gp120-gp41 trimer, where the antibody is accessible. It shows the main areas that can be done.

FIG. 2. Co-transfection of furin increases the efficiency of cleavage of the peptide bond between gp120 and gp41: In 293T cells in the presence or absence of a plasmid expressing co-transfected furin, HIV-1 JR-FL gp140WT, or g
DNA expressing the p140UNC (gp120-gp41 cleavage site mutant) protein was transfected. The 35S-labeled envelope glycoprotein secreted from the cells was immunoprecipitated with anti-gp120 MAb 2G12 and then analyzed by SDS-PAGE. Lane 1: gp140WT (gp140 / g
p120 double line); lane 2: gp140WT + furin (gp120 only); lane 3: gp140UNC (gp140 only); lane 4: gp140U.
NC + furin (gp140 only). Approximate molecular weight of major molecular species (kDa
) Is shown on the left.

FIG. 3A: Positions of cysteine substitutions in JR-FL gp140: a) Various diagrams of cysteine-mutated JR-FL gp140WT proteins in one or more mutants on a schematic diagram of JR-FL gp120 gp120 and gp41 ECTO subunits. The residues are indicated by black arrows. The positions of the alanine 492 and threonine 596 residues, both mutated to cysteine, in the SOS gp140 protein are indicated by the larger solid arrow. C-terminal of gp120 and N of gp41
The open square at the end indicates the region in the gp140UNC protein that was mutated to remove the cleavage site between gp120 and gp41.

FIG. 3B: Positions of cysteine substitutions in JR-FL gp140: b) Schematic diagrams of JR-FL gp41 gp120 and gp41 ECTO subunits showing various cysteine-mutated JR-FL gp140WT proteins in one or more mutants. The residues are indicated by black arrows. The positions of the alanine 492 and threonine 596 residues, both mutated to cysteine, in the SOS gp140 protein are indicated by the larger solid arrow. C-terminal of gp120 and N of gp41
The open square at the end indicates the region in the gp140UNC protein that was mutated to remove the cleavage site between gp120 and gp41.

FIG. 4: Immunoprecipitation analysis of selected double cysteine variants of JR-FL gp140: 35S-labeled envelope glycoproteins secreted from transfected 293T cells were immunoprecipitated with anti-gp120 and anti-gp41 MAbs. , And then analyzed by SDS-PAGE. The MAb used was 2G12 (anti-gp120 C
3-V4 region) or F91 (anti-gp120 CD4 binding site region). The positions of the two cysteine substitutions in each protein (one in gp120 and the other in gp41ECTO) are noted above the lanes. The gp140WT protein is shown in lane 15. All proteins except the gp140WT protein were expressed in the presence of co-transfected furin.

Figure 5: The efficiency of intermolecular disulfide bond formation depends on the position of cysteine substitution: Secreted from 293T cells cotransfected with furin 35
Enveloped glycoproteins labeled with S and the various gp140 variants described above were immunoprecipitated with anti-gp120 MAb 2G12, followed by SDS-PAG.
It analyzed by E. For each mutant, the intensities of the 140 and 120 kDa bands were determined by densitometry and the gp140 / gp140 + gp120 ratio was calculated and recorded. The degree of shading is proportional to the magnitude of the ratio of gp140 / gp140 + gp120. Position of amino acid substitution in gp41, and C1 and C of gp120
The positions of the amino acid substitutions in the 5 domains are listed above and below the sides, respectively. N. D. = Not implemented.

FIG. 6: Confirmation that intermolecular bonds of gp120 and gp41 are formed in the SOS gp140 protein: Using a plasmid expressing the gp140 protein (a plasmid expressing furin, if specifically mentioned), 293T cells were transfected. Secreted 35S-labeled glycoproteins were immunoprecipitated using the indicated MAbs and analyzed by SDS-PAGE under reducing (+ DTT) or non-reducing conditions. A. 2 of SOSgp140, gp140WT, and gp140UNC proteins
Radioimmunoprecipitation with G12. The immunoprecipitated protein was subjected to reducing conditions (lane 4 to
6) or under non-reducing conditions (lanes 1-3), separated by SDS-PAGE. B. SOS gp140 protein containing the corresponding single cysteine mutation and g
Radioimmunoprecipitation of p140 protein with 2G12. The 140 kDa protein band is not observed in either the A492C or T596C single cysteine mutant gp140 proteins. C. Radioimmunoprecipitation with 2G12 of SOS gp140 protein produced in the presence or absence of co-transfected furin. The immunoprecipitated proteins were subjected to reducing conditions (lanes 3-4) or non-reducing conditions (lanes 1-2).
Separation by SDS-PAGE. In the presence of exogenous furin, DT
It has been shown that T attenuates the produced 140 kDa SOS protein band, but not in the absence of exogenous furin.

Figure 7. Analysis of cysteine mutants of JR-FL gp140: 35s-labeled envelope glycoprotein secreted from transfected 293T cells was immunoprecipitated with anti-gp120 MAb 2G12 followed by SD.
It was analyzed by S-PAGE. All gp140 were expressed in the presence of co-transfected furin. Lanes 1-8: gp140 containing the indicated double cysteine mutation. Lanes 9-11: A492C / T596C with the indicated lysine to alanine substitutions present at residue 491 (lane 9), residue 493 (lane 10), or both residues 491 and 493 (lane 11). A gp140 protein containing a double cysteine substitution. Lanes 12-14: g with quadruple cysteine substitution
p140 protein.

FIG. 8 SOS gp140, W44C / T596C gp140 mutant, gp140
Comparison of antigenic structures of UNC and gp140WT proteins: 35S-labeled envelope glycoproteins secreted from transfected 293T cells were immunoprecipitated with the indicated anti-gp120 MAbs and anti-gp41 MAbs followed by SDS-PAGE. analyzed. Mutant gp140 was expressed in the presence of co-transfected furin, but not wild-type gp140. A. An anti-gp120 immunoglobulin that neutralizes HIV-1 _JR-FL . B. Non-neutralizing antibody to the C1, C4 and C5 regions of gp120. C. Antibodies to the CD4 derived epitopes were investigated alone and in combination with sCD4. D. Neutralized (2F5) and non-neutralized (7B2, 2.2B and 25C2) anti-gp41
Antibody and MAb 2G12. E. Radioimmunoprecipitation of gp140WT (odd lanes) and gp140UNC (even lanes).

FIG. 9: gp1 from various HIV-1 isolates stabilized by disulfide bonds
Preparation of 40 proteins: As shown in the figure, in the presence or absence of exogenous furin,
Plasmids expressing wild type or mutant gp140 were transfected into 293T cells. The supernatant labeled with 35S was prepared and MAb 2G12 was prepared as described above.
It was analyzed by radioimmunoprecipitation at. Lane 1: SOS gp140 protein. Lane 2: gp140WT + furin. Lane 3: gp140WT, no furin. (A) HIV-1 DH123. (B) HIV-1 HxB2.

FIG. 10-1: Amino acid sequence of glycoprotein with various deletions in the variable region. The deleted wild-type sequence is shown in white shadow and includes the following: ΔV1: D132-K
152; ΔV2: F156 to I191.

FIG. 10-2: Amino acid sequence of glycoprotein with various deletions in the variable region. The deleted wild-type sequence is shown in white shadow and includes: ΔV1V2 ′: D13
2 to K152 and F156 to I191; ΔV1V2 *: V126 to S192; Δ
V3: N296 to Q324.

FIG. 11. Formation of intersubunit cysteine bridges in envelope proteins with deletions in the variable loop region. a) ΔV1V2 * V3 protein with two cysteines at positions 492 and 596 (indicated by CC) and ΔV1V2 * V3 N
The 357Q N398Q protein was precipitated with 2G21 and F91 (lanes 3 and 7, and 4 and 8, respectively). Suitable controls without the cysteine mutation are shown in lanes 1, 2, 5 and 6. Wild-type protein without extra cysteine is shown in lanes 9 and 10. All proteins except the wild type protein in lane 10 were cleaved by furin. The approximate size is shown in kDa on the right. b) Various loop deletion proteins with two cysteines (CC) at positions 492 and 596 were precipitated with 2G12 (lanes 3, 5, 7, 9, 11 and 13). Proteins with identical deletions without extra cysteines are shown in adjacent lanes. These control proteins were not cleaved by furin. Full length SOS gp14
0 protein is included in lane 1 as a control.

FIG. 12. Antigenic characterization of the A492C / T596C mutant with a deletion in the variable loop. All variants were expressed in the presence of exogenous furin. The antibody used in RIPA is shown at the top. a) A492C / T596C ΔVLV2 *
And b) A492C / T596C ΔV3 mutant.

FIG. 13: Nucleotide (A) and amino acid (B) sequence of HIV-1 _JR-FL SOS gp140. Amino acid numbers correspond to those of wild type JR-FL (Genba
nk reception number U63632). Cysteine mutations are shown in bold and underlined.

FIG. 14: Nucleotides of HIV-1 _JR-FL ΔV1V2 * SOS gp140 (A
) And amino acid (B) sequences. The amino acid numbers correspond to those of wild type JR-FL (Genbank Accession No. U63632). Cysteine mutations are shown in bold and underlined.

FIG. 15: Nucleotide (A) and amino acid (B) sequences of HIV-1 _JR-FL ΔV3 SOS gp140. The amino acid numbers correspond to those of wild type JR-FL (Ge
nbank reception number U63632). Cysteine mutations are shown in bold and underlined.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61P 31/18 C07K 14/16 4C086 C07K 14/16 16/10 4H045 16/10 19/00 19/00 C12N 1/15 C12N 1/15 1/19 1/19 1/21 1/21 7/00 5/10 C12P 21/08 7/00 C12N 15/00 ZNAA // C12P 21/08 5/00 A (72) Inventor Binley, James M. New York, USA 11217 Wyckoff Street, Brooklyn 141 (72) Inventor Schuerke, Norbert USA, New York 10596 New City, Ridge Road 101 (72) Inventor Olson, William Sea USA , Phone Court 21 (72), New York, 10562 Ossining Madon, Paul Jay, New York, USA 10583 Scarsdale, Fox Meadow Road 191 (72) Inventor Moore, John Peay United States, New York 10021 New York, East Sixth Sud Street 500 F-term (reference) 4B024 AA01 AA14 BA32 BA51 CA04 CA07 CA11 DA01 DA02 DA05 DA11 EA01 EA02 EA03 EA04 4B064 AG27 CA10 CA20 CC24 DA01 DA15 4B065 AA01X AA57X AA87X AA95X AA95Y AB01 AB02 BA14 ZA33A14C14 CA14 4A0B14C17 CA14 CA14 4A0A4A17B02 CA14 CA14 4A0A3 BB41 BB43 BB44 CC32 EE01 GG01 4C086 AA01 AA02 EA16 MA01 MA04 NA14 ZB33 ZC55 4H045 AA10 AA11 AA20 AA30 BA10 BA41 CA05 DA76 DA86 EA31 EA53 FA72 FA74

Claims (86)

[Claims] 1. An isolated nucleic acid comprising a nucleotide moiety having a sequence encoding a complex comprising a viral surface protein and a corresponding viral transmembrane protein, said complex being one of the amino acid sequences. An isolated nucleic acid comprising the above mutation, wherein the mutation enhances the stability of the complex formed between the surface protein of the virus and the transmembrane protein of the virus. 2. The isolated nucleic acid of claim 1, wherein the virus is a lentivirus. 3. The isolated nucleic acid of claim 1, wherein the virus is human immunodeficiency virus. 4. The human immunodeficiency virus is a primary isolate.
). The isolated nucleic acid of claim 3, which is 5. The human immunodeficiency virus is HIV-1 _JR-FL , HIV-1 _DH123 or HIV.
The isolated nucleic acid according to claim 3, which is -1 _Gun-1 , HIV-1 _89.6 or HIV-1 _HXB2 . 6. The isolated nucleic acid of claim 3, wherein the viral surface protein is gp120 or a modified version of gp120 and the modification alters the immunogenicity of a molecule associated with wild-type gp120. 7. The isolated nucleic acid of claim 6, wherein the modified gp120 molecule is characterized by the lack of one or more variable loops present in wild-type gp120. 8. The isolated nucleic acid of claim 7, wherein the variable loop comprises V1, V2 or V3. 9. The any of claims 6-8, wherein the modified gp120 molecule is characterized by the absence (or presence) of one or more standard glycosylation (s) present (or absent) in wild type gp120. The isolated nucleic acid of paragraph 1. 10. The isolated nucleic acid of claim 9, wherein one or more canonical glycosylation sites are missing from the V1V2 region of the gp120 molecule. 11. The isolation of any of claims 3-10, wherein the transmembrane protein is gp41 or a modified version of gp41 and the modification alters the immunogenicity of a molecule associated with wild type gp41. Nucleic acid. 12. The transmembrane protein comprises the gp41 ectodomain (ectodom).
12. The isolated nucleic acid of claim 11, which is ain). 13. The transmembrane protein of claim 11, wherein the transmembrane protein is modified by the absence (or presence) of one or more canonical glycosylation sites that are absent (or present) in the wild-type gp120. Isolated nucleic acid. 14. Stabilization of said complex is achieved by one or more cysteine-cysteine bonds formed between said superficial and transmembrane proteins, said bonds being present in said corresponding wild-type complex. No, any one of claims 1 to 13
An isolated nucleic acid according to paragraph. 15. The isolation of claim 14, wherein one or more amino acids adjacent to the introduced cysteine, or one or more amino acids containing atoms within 5 angstroms of said cysteine, are mutated to non-cysteine residues. Nucleic acid. 16. One or more cysteines in gp120 or a modified version of gp120 are:
disulfide-bonded to one or more cysteines of gp41 or gp41-modified,
16. The isolated nucleic acid of claim 14 or 15. 17. A cysteine in the C5 region of gp120 or a modified gp120 is
A disulfide bond to the cysteine of the ectodomain of gp41.
The isolated nucleic acid according to 16. 18. The disulfide bond has an A492C mutation in gp120 and T5 in gp41.
17. The isolated nucleic acid of claim 16, formed between cysteines introduced by the 96C mutation. 19. The isolated nucleic acid molecule of claim 1, which is a cDNA. 20. The isolated nucleic acid molecule of claim 1, which is genomic DNA. 21. The isolated nucleic acid molecule of claim 1, which is RNA. 22. A replicable vector comprising the nucleic acid of claim 1. 23. A plasmid, cosmid, or λ containing the nucleic acid according to claim 1.
Phage or YAC. 24. The plasmid of claim 23, designated PPI4. 25. A host cell comprising the vector of claim 22. 26. The cell of claim 25, wherein the cell is a eukaryotic cell. 27. The cell of claim 25, wherein the cell is a bacterial cell. 28. A vaccine comprising the isolated nucleic acid of claim 1. 29. A vaccine comprising a therapeutically effective amount of the nucleic acid of claim 1. 30. A vaccine comprising a therapeutically effective amount of the protein encoded by the nucleic acid of claim 1. 31. A method of treating a viral disease, comprising treating a subject infected with a virus according to claim 29 or 30 or a combination thereof by treating the subject. 32. A vaccine comprising a prophylactically effective amount of the nucleic acid of claim 1. 33. A vaccine comprising a prophylactically effective amount of a protein encoded by the nucleic acid of claim 1. 34. A method of reducing the likelihood of a subject being infected with a virus, wherein the subject is infected with the virus by administration of the vaccine of claim 32 or 33 or a combination thereof. A method comprising reducing the likelihood. 35. A vaccine comprising the nucleic acid according to any one of claims 3-18. 36. A vaccine comprising a therapeutically effective amount of the nucleic acid of any one of claims 3-18. 37. A vaccine comprising a therapeutically effective amount of a protein encoded by the nucleic acid of any one of claims 3-18. 38. A method of treating an HIV-1-infected subject, wherein the subject is immunized with the vaccine of claim 36 or 37 or a combination thereof. A method including: 39. A vaccine comprising a prophylactically effective amount of the nucleic acid of any one of claims 3-18. 40. A vaccine comprising a prophylactically effective amount of the protein encoded by the nucleic acid according to any one of claims 3-18. 41. A method of reducing the likelihood of being infected with HIV-1 in a subject, comprising administering the vaccine of claim 39 or 40 or a combination thereof to the subject. A method comprising reducing the likelihood of being infected with HIV-1. 42. The vaccine is a recombinant subunit protein, DNA
36. The vaccine of claim 35, which comprises a plasmid, a replicating viral vector, a non-replicating viral vector or a combination thereof. 43. A method of reducing the severity of HIV-1 disease in a subject, comprising:
In subsequent exposure to HIV-1, by administering the vaccine of claim 39 or 40 or a combination thereof prior to exposure of the subject to IV-1.
A method comprising reducing the severity of HIV-1 disease or AIDS in said subject. 44. A viral envelope protein comprising a viral surface protein and a corresponding viral transmembrane protein, wherein the viral envelope protein comprises one or more mutations in the amino acid sequence, the mutations being the surface layer of the virus. A viral envelope protein that enhances the stability of the complex formed between the protein and the transmembrane protein. 45. A complex comprising a viral surface protein and a viral transmembrane protein, wherein the complex comprises one or more mutations in the amino acid sequence, wherein the mutation is the viral surface protein and the transmembrane protein. A complex that enhances the stability of the complex formed between proteins. 46. A mutated HIV-1 envelope protein encoded by the nucleic acid of any one of claims 3-18. 47. The protein of claim 44 or the complex of claim 45, which is linked to at least one other protein or protein fragment to form a fusion protein. 48. The purified protein according to any one of claims 44-46. 49. A vaccine comprising a therapeutically effective amount of the protein of claim 44 or the complex of claim 45. 50. A vaccine comprising a prophylactically effective amount of the protein of claim 44 or the complex of claim 45. 51. A protein or a protein according to claim 44 in a subject
A method for stimulating or enhancing the production of an antibody that recognizes the complex according to 45. 52. An antibody, antibody chain, fragment or derivative thereof which is isolated or identified using the viral envelope protein encoded by the recombinant nucleic acid of claim 1. 53. The antibody of claim 52, wherein the antibody is of the IgM, IgA, IgE or IgG class or a subclass thereof. 54. The antibody fragment of claim 52, including but not limited to Fab, Fab '(Fab') 2, Fv and single chain antibodies. 55. An isolated antibody light chain of the antibody of claim 52, or a fragment or oligomer thereof. 56. An isolated antibody heavy chain of the antibody of claim 52, or a fragment or oligomer thereof. 57. One or more complementarity determining regions of the antibody of claim 52. 58. The antibody of claim 52, which is derivatized in such a manner as by the addition of an affinity ligand such as a fluorescent functional group, a radionuclide, an enzyme, a toxin or biotin. 59. The antibody of claim 52, wherein the antibody is a human antibody. 60. The method of claim 52 or 59, wherein the antibody is a monoclonal antibody.
The antibody according to. 61. The antibody of claim 52, wherein the antibody is a humanized antibody. 62. The antibody of claim 52 or any one of claims 59-61, wherein the viral envelope protein is derived from HIV-1. 63. An isolated nucleic acid molecule encoding the antibody of any one of claims 52 or 59-61, wherein said nucleic acid molecule is RNA, genomic DNA or
A nucleic acid molecule that is a cDNA. 64. The isolated nucleic acid of claim 63, wherein the viral envelope protein is from HIV-1. 65. An agent capable of inhibiting the binding of the antibody according to claim 52. 66. A method of reducing the likelihood of HIV-1 exposure of a subject exposed to HIV-1, comprising the antibody of claim 62 or the isolated of claim 64. A method comprising reducing the likelihood of HIV-1 exposed subjects being infected with HIV-1 by administering a nucleic acid. 67. A method of treating a subject infected with HIV-1, comprising:
65. A method comprising treating the subject by administering the antibody of claim 2 or the isolated nucleic acid of claim 64. 68. An agent capable of binding to the envelope protein of the mutant virus encoded by the recombinant nucleic acid molecule according to claim 1. 69. The agent of claim 68, which inhibits viral infection. 70. The agent of claim 69, wherein the viral envelope protein is derived from HIV-1. 71. A method for determining whether a compound is capable of inhibiting a viral infection, comprising: (A) an appropriate concentration of said compound and encoded by said nucleic acid of claim 1. Contacting the mutant protein envelope protein with the protein under conditions permitting binding of the compound; (B) the complex and reporter molecule resulting from step (A) above; Contacting under conditions that permit binding of the reporter molecule to the mutated viral envelope protein in the absence of the compound; (C) determining the amount of bound reporter molecule; and (D) Comparing the amount of bound reporter molecule in step (c) with the amount determined in the absence of said compound, said reduction of said amount being Method shown can inhibit infection by the virus. 72. The reporter molecule is an antibody or derivative thereof.
The method described in 71. 73. The method of claim 71, wherein the reporter molecule comprises one or more host cell virus receptors or molecular mimetics thereof. 74. A method of determining whether a compound is capable of inhibiting a viral infection, comprising: (a) providing said compound in a suitable concentration and a host cell viral receptor or a molecular mimic thereof Contacting the compound in the absence of the compound under conditions permitting binding of the receptor or a mimetic of the receptor; (b) the complex resulting from step (A) above; Item 1, wherein the mutant viral envelope protein encoded by the recombinant nucleic acid according to Item 1 is allowed to bind to the envelope protein and the receptor or a mimetic of the receptor in the absence of the compound. Contacting under; (c) measuring the amount of binding of the envelope protein to the receptor or a mimetic of the receptor; (d) the binding determined in step (c) Comparing the amount to the amount determined in the absence of the compound, the reduction in the amount indicating that the compound is capable of inhibiting infection by the virus. 75. The method of any of claims 71-74, wherein the virus is HIV-1. 76. The method of claim 71 or 72, wherein the host cell viral receptor is CD4, CCR5, CXCR4 or combinations or molecular mimetics thereof. 77. The method of any one of claims 71-76, wherein said compound was previously unknown. 78. A compound determined to be capable of inhibiting viral infection by the method of any one of claims 71-76. 79. An amount of a compound effective to inhibit a viral infection determined to be capable of inhibiting a viral infection by the method of any one of claims 71-76, and a pharmaceutical A pharmaceutical composition comprising an acceptable carrier. 80. The pharmaceutical composition of claim 79, wherein the viral infection is HIV-1 infection. 81. A viral envelope protein comprising a viral surface protein and a corresponding viral transmembrane protein, wherein said viral envelope protein is formed between said viral surface protein and transmembrane protein. A virus envelope protein comprising one or more mutations of an amino acid sequence that enhances stability, wherein the surface layer protein and the transmembrane protein are encoded by different nucleic acids. 82. A complex comprising a viral surface protein and a corresponding viral transmembrane protein of a viral envelope protein, wherein said viral envelope protein is formed between said viral surface protein and transmembrane protein. A complex comprising one or more mutations in an amino acid sequence that enhances body stability, wherein the surface protein and the transmembrane protein are encoded by different nucleic acids. 83. An antibody that binds to the protein of claim 44 or the complex of claim 45, but does not cross-react with individual monomeric surface proteins or individual monomeric transmembrane proteins. 84. The antibody of claim 83, which is capable of binding the HIV-1 virus. 85. A virus-like particle comprising the complex of claim 45. 86. The method further comprising an immunodeficiency virus gag protein.
Virus-like particles according to 85.