Method for Removing Immunosuppressive Properties of HIV Envelope Glycoproteins

The present invention relates to removal of immunosuppressive effects of envelope glycoproteins derived from human immunodeficiency virus for vaccination purposes and for generation of neutralizing antibodies to HIV.

Technical Background

There are two major types of HIV; HIV-1 and HIV-2 that infect humans. SIV is a virus very similar to HIV which infects monkeys and has been found in African monkeys. They all belong to a group of retroviridae known as Lentiviruses. Retroviruses transfer their genes from a producer cell to a target cell as a genomic RNA transcript, which is reverse-transcribed after infection and integrated into the DNA genome of the target cell.

The first person with a documented HIV-infection died in 1959. In the early 1980s doctors in the US become aware that more and more patients suffered from abnormal infections and showed signs of immune failure. The syndrome was named Acquired Immune Deficiency Syndrome (AIDS) and it was soon after discovered that HIV was the causative agent for the observed destruction of the immune system.

Only attenuated (that is live but weakened) HIV-strains has been able to provide immunity in primate studies even though they will never reach a required safety profile suitable for mass vaccination. The replication process for HIV-1 has an error rate of about one per 10,000 base pairs. Since the entire viral genome is just under 10,000 base pairs, it is estimated that on average one error is introduced into the HIV-1 genome at each viral replication cycle. This high mutation rate contributes to extensive variability of the viruses inside anyone person and an even wider variability across populations. This variability has resulted in different HIV-1 variants being described, where these subspecies of virus are called “clades.” The distinctions are based on the structure of the envelope proteins, which are especially variable. The M (for major) variant is by far the most prevalent worldwide. Within the M variant are clades A, B, C, D, E, F, G H, I, J and K, with clades A through E representing the vast majority of infections globally. Clades A, C and D are dominant in Africa, while clade B is the most prevalent in Europe, North and South America and Southeast Asia. Clades E and C are dominant in Asia. These clades differ by as much as 35%. Another variant is Clade 0, which is observed in Cameroun isolates of HIV-1. The greatest variation in structure is seen in the envelope proteins gpl20 and gp41. There are two important results from the very high mutation rate of HIV-1 that have profound consequences for the epidemic. First, the high mutation rate is one of the mechanisms that allow the virus to escape from control by drug therapies. These new viruses represent resistant strains. The high mutation rate also allows the virus to escape the patient's immune system by altering the structures that are recognized by immune components. An added consequence of this extensive variability is that the virus can also escape from control by vaccines.

SUMMARY OF THE INVENTION

Envelope glycoproteins derived from HIV with reduced immunosuppressive properties are antigens, which may have one or more of the following uses:

I) Providing a strong and persistent CD8⁺ T-cell response against HIV-virus glycoprotein in vivo

II) Generating neutralizing antibodies against HIV-virus in vivo

III) Providing an optimal antigen for vaccination against HIV

IV) Use as an antigen for generation of neutralizing antibodies against HIV

The strategy is based upon eliminating the inherent immunosuppressive properties of the envelope glycoprotein of HIV without disrupting the overall conformation of the envelope. This will be accomplished by rational introduction of specific amino acids changes into the HIV envelope glycoprotein.

The present invention relates to HIV-1 envelope polypeptides, to be used either as an antigen for HIV vaccination or as an antigen for generation of neutralizing antibodies against HIV. The invention also encompasses biological entities comprising such polypeptides or nucleic acids encoding those, in particular viral particles, viral like particles whether they are produced in vivo or in vitro, viral derived vector(s) or any eukaryotic expression vector(s). Moreover the invention relates to vaccine compositions, which comprise a polypeptide of the present invention and an adjuvant as well as a production method and kits comprising said vaccine compositions.

General Disclosure
Vaccination

Vaccination is the administration of antigenic material (a vaccine) to produce immunity to a disease.

Mechanism of Function

Vaccinations involve the administration of one or more immunogens, which can be administered in several forms.

Some vaccines are administered after the patient already has contracted a disease. Vaccinia given after exposure to smallpox, within the first three days, is reported to attenuate the disease considerably, and vaccination up to a week after exposure likely offers some protection from disease or may modify the severity of disease. Other examples include experimental AIDS, cancer and Alzheimer's disease vaccines.

Adjuvants and Preservatives

Vaccines typically contain one or more adjuvants, used to boost the immune response. Tetanus toxoid, for instance, is usually adsorbed onto alum. This presents the antigen in such a way as to produce a greater action than the simple aqueous tetanus toxoid.

Vaccines may further comprise preservatives, which are used to prevent contamination with bacteria or fungi. Preservatives may be used at various stages of production of vaccines.

Types

All vaccinations work by presenting a foreign antigen to the immune system in order to evoke an immune response, but there are several ways to do this. The four main types that are currently in clinical use are as follows:

- 1. An inactivated vaccine consists of virus particles which are grown in culture and then killed using a method such as heat or formaldehyde. The virus particles are destroyed and cannot replicate, but the virus capsid proteins are intact enough to be recognized and remembered by the immune system and evoke a response. When manufactured correctly, the vaccine is not infectious, but improper inactivation can result in intact and infectious particles. Since the properly produced vaccine does not reproduce, booster shots are required periodically to reinforce the immune response.
- 2. In an attenuated vaccine, live virus particles with very low virulence are administered. They will reproduce, but very slowly. Since they do reproduce and continue to present antigen beyond the initial vaccination, boosters are required less often. These vaccines are produced by passaging virus in cell cultures, in animals, or at suboptimal temperatures, allowing selection of less virulent strains, or by mutagenesis or targeted deletions in genes required for virulence. There is a small risk of reversion to virulence, this risk is smaller in vaccines with deletions. Attenuated vaccines also cannot be used by immune-compromised individuals.
- 3. Virus-like particle vaccines consist of viral protein(s) derived from the structural proteins of a virus. These proteins can self-assemble into particles that resemble the virus from which they were derived but lack viral nucleic acid, meaning that they are not infectious. Because of their highly repetitive, multivalent structure, virus-like particles are typically more immunogenic than subunit vaccines (described below). The human papillomavirus and Hepatitis B virus vaccines are two virus-like particle-based vaccines currently in clinical use.
- 4. A subunit vaccine presents an antigen to the immune system without introducing viral particles, whole or otherwise. One method of production involves isolation of a specific protein from a virus or bacteria (such as a bacterial toxin) and administering this by itself. A weakness of this technique is that isolated proteins may have a different three-dimensional structure than the protein in its normal context, and will induce antibodies that may not recognize the infectious organism. In addition, subunit vaccines often elicit weaker antibody responses than the other classes of vaccines.
  
  A number of other vaccine strategies are under experimental investigation. These include DNA vaccination and recombinant viral vectors.

Superiority of Virus Like Particles as Immunogens for Vaccination Purposes or for Generation of Neutralizing Anti Bodies

The development of a broad and neutralizing antibody response is vital for a protective HIV-1 vaccine. In addition, the induction of specific and effective cytotoxic T lymphocytes has been shown to be required for infection control. Thus, the developments of vaccine strategies that encompass both arms of the immune system are important. Differential MHC I and II antigen presentation is a key factor for initiation of a potent immune response. There appear to be short-comings in antigen presentation when antigens are administered solely as peptides or DNA. In contrast cross-talk between antigen presenting cells and T helper lymphocytes are promoted in vaccine strategies based on either infectious or non-infectious viral particles (virus like particles or VLPs). A potent induction of cell mediated immunity can be achieved even with whole-killed viral particles. This is likely a consequence of improved antigen uptake and systemic immunostimulation in macrophages and dendritic cells. Baculovirus-derived human immunodeficiency virus type i virus-like particle activate dendritic cels and induce ex vivo t-cell responses). To achieve potent immunogenic retroviral particles such as γ-retroviral particles or lentiviral like particles may be produced with HIV-1 envelope trimers on the surface. These particles can function as a superior immunogenic particle avoiding any risk associated with viral inactivation procedures. In a further development such VLPs can be produced by cells in the patient using appropriate vectors like but not restricted to vectors derived from vaccinia virus or measles virus

Administration

A vaccine administration may be by any acceptable route, such as oral, by injection (intramuscular, intradermal, subcutaneous), by puncture, transdermal or intranasal.

Vaccinia Virus (VACV or VV)

Poxviruses are unique among DNA virus because they replicate only in cytoplasm of the host cell, outside of the nucleus. Therefore, the large genome is required for encoding various enzymes and proteins involved in viral DNA replication and gene transcription. During its replication cycle, VV produces four infectious forms which differ in their outer membranes: intracellular mature virion (IMV), the intracellular enveloped virion (IEV), the cell-associated enveloped virion (CEV) and the extracellular enveloped virion (EEV). Although the issue remains contentious, the prevailing view is that the IMV consists of a single lipoprotein membrane, while the CEV and EEV are both surrounded by two membrane layers and the IEV has three envelopes. The IMV is the most abundant infectious form and is thought to be responsible for spread between hosts. On the other hand, the CEV is believed to play a role in cell-to-cell spread and the EEV is thought to be important for long range dissemination within the host organism.

Common Strains

This is a list of some of the well-characterized vaccinia strains used in research and immunizations.

- Western Reserve
- Copenhagen
- Dryvax (also known as “Wyeth”): the vaccine strain previously used in the United States, produced by Wyeth. It was replaced in 2008 by ACAM2000, produced by Acambis. It was produced as preparations of calf lymph which was freeze-dried and treated with antibiotics.
- ACAM2000: The current strain in use in the USA, produced by Acambis. ACAM2000 was derived from a clone of a Dryvax virus by plaque purification. It is produced in cultures of Vero cells.
- Modified vaccinia Ankara: a highly attenuated (not virulent) strain created by passaging vaccinia virus several hundred times in chicken embryo fibroblasts. Unlike some other vaccinia strains it does not make immunodeficient mice sick and therefore may be safer to use in humans who have weaker immune systems due to being very young, very old, having HIV/AIDS, etc.

Vaccinia viruses re-engineered to express foreign genes are robust vectors for production of recombinant proteins, the most common being a vaccine delivery system for antigens. Concerns about the safety of the vaccinia virus have been addressed by the development of vectors based on attenuated vaccinia viruses. One of them, the Modified Vaccinia Ankara (MVA) virus, is a highly attenuated strain of vaccinia virus that was developed towards the end of the campaign for the eradication of smallpox by Professor Anton Mayr in Germany. Produced by hundreds of passages of vaccinia virus in chicken cells, MVA has lost about 10% of the vaccinia genome and with it the ability to replicate efficiently in primate cells.

Modified Vaccinia Ankara in Clinical Trials

MVA is widely considered as the vaccinia virus strain of choice for clinical investigation because of its high safety profile. MVA has been administered to numerous animal species including Monkeys, mice, swine, sheep, cattle, horses, and elephants, with no local or systemic adverse effects. Over 120,000 humans have been safely and successfully vaccinated against smallpox with MVA by intradermal, subcutaneous, or intramuscular injections.

Challenge Studies in Primates

Immunization regimens incorporating priming with DNA vaccine and boosting with recombinant MVA-based vaccine have been found to provide some protection in non-human primates following challenge with an immunodeficiency virus. While vaccination did not prevent infection in these studies, it did result in lower viral load setpoints, increased CD4 counts, and reduced morbidity and mortality in vaccinated animals, compared to controls.

Production of a Lentiviral VLPs by an MVA Vaccine in Vivo

A modified vaccinia virus Ankara (MVA) vaccine has been used in the SIV macaque therapeutic immunization model. MVA was selected as a vaccine vector, since it has previously shown to induce strong antiviral immune responses and protection. As viral antigens SIV Gag-Pol and Env were used.

Neutralizing Antibody

A Neutralizing antibody, or NAb is an antibody which defends a cell from an antigen or infectious body by inhibiting or neutralizing any effect it has biologically. An example of a neutralizing antibody is diphtheria antitoxin, which can neutralize the biological effects of diphtheria toxin.

Most antibodies work by binding to an antigen, signaling to a white blood cell that this antigen has been targeted, after which the antigen is processed and consequently destroyed. The difference between neutralizing antibodies and binding antibodies is that neutralizing antibodies neutralize the biological effects of the antigen, while binding antibodies flag antigens.

This difference is what gives neutralizing antibodies the ability to fight viruses which attack the immune system, since they can neutralize function without a need for white blood cells (excluding production)

Immunosuppressive Properties of Enveloped Viruses with Type I Fusion Proteins

Enveloped viruses enter their target cells by means of active fusion of their lipid membrane with that of the cells. The fusion is mediated by viral gene products in form of proteins embedded in the lipid membrane of the virus, the so-called viral fusion proteins [1].

At the same time fusion proteins of some enveloped viruses show an immune suppressive activity. Inactivated retroviruses are able to inhibit proliferation of immune cells upon stimulation [2-4]. Expression of these proteins is enough to enable allogenic cells to grow to a tumor in immune competent mice. In one study, introduction of ENV expressing construct into MCA205 murine tumor cells, which do not proliferate upon s.c. injection into an allogeneic host, or into CL8.1 murine tumor cells (which overexpress class I antigens and are rejected in a syngeneic host) resulted in tumor growth in both cases [5]. Such immunosuppressive domains have been found in a variety of different viruses with type 1 fusion mechanism such as Mason pfeizer monkey virus (MPMV), murine leukemia virus (MLV), lentiviruses such as HIV and in filoviruses such as Ebola and Marburg viruses [6-9].

The immune suppressive activity is located to a very well-defined structure within the class I fusion proteins. The immunosuppressive effects range from significant inhibition of lymphocyte proliferation [7,8], cytokine skewing (up regulating IL-10; down regulating TNF-α, IL-12, IFN-γ) [10] and inhibition of monocytic burst [11] to cytotoxic T cell killing [12]. Importantly, peptides spanning ISD are mostly active in these assays when either linked as dimers or coupled to a carrier (i.e. >monomeric). Such peptides derived from such immune suppressive domains are able to reduce or abolish immune responses such as cytokine secretion or proliferation of T-cells upon stimulation.

Co-Location of the Immunosuppression Domain and the Fusion Domain

The immunosuppressive domain of retro-, lenti- and filoviruses overlap a structurally important part of the fusion subunits of the envelope proteins. Although the primary structure (sequence) of this part of the fusion proteins can vary greatly from virus to virus, the secondary structure, which is very well preserved among different virus families, is that of an alpha helix that bends in different ways during the fusion process This structure plays a crucial role during events that result in fusion of viral and cellular membranes. It is evident that the immunosuppressive domains of the class I fusion proteins overlap with a very important protein structure needed for the fusion proteins mechanistic function.

The energy needed for mediating the fusion of viral and cellular membranes is stored in the fusion proteins, which are thus found in a meta-stable conformation on the viral surface. Once the energy is released to drive the fusion event, the protein will find its most energetically stable conformation. In this regard fusion proteins can be compared with loaded springs that are ready to be sprung. This high energy conformation makes the viral fusion proteins very susceptible to modifications; Small changes in the primary structure of the protein often result in the protein to be folded in its stable post fusion conformation. The two conformations present very different tertiary structures of the same protein.

This presents a significant challenge if one wishes to remove the immunosuppressive effect without altering the tertiary structure of the fusion protein, but it has been shown in the case of simple retroviruses that small structural changes in the envelope protein are sufficient to remove the immune suppressive effect without changing structure and hence the antigenic profile, resulting in an antigen which is significantly more potent as a vaccine.

The non-immune suppressive envelopes are much better antigens for vaccination. The proteins can induce a 30-fold enhancement of anti-env antibody titers when used for vaccination [6]. Furthermore, viruses that contain the non-immunosuppressive form of the friend murine leukemia virus envelope protein, although fully infectious in irradiated immunocompromised mice cannot establish an infection in immunocompetent animals. Interestingly in the latter group the non-immunosuppressive viruses induce higher cellular and humoral immune responses, which fully protect the animals from subsequent challenge by wild type viruses [13]

Further General Description of the Invention

The present invention provides a new approach to designing immunogenic HIV-1 envelope polypeptides derived from HIV-1 envelope protein, GP41 and/or GP120 and/or GP160 for vaccination purposes and/or for generation of neutralizing antibodies to HIV. Specific amino acid residues are mutated to repress the immunosuppression displayed by GP41 thereby boosting the immune response against HIV-1. Mutation of those specific residues however, must not completely destroy the overall protein structure of the viral envelope protein as e.g. measured functionally by fusion assays. The polypeptides of the present invention can be inserted in a suitable construct and expressed in an organism to produce a vaccine or an immunogenic response against HIV.

According to an aspect, the present invention concerns a method for making an HIV-1 envelope glycoprotein, which may be expressed on the surface of cells, virus or virus like particles, and which provides reduced immune suppression, allowing said HIV-1 envelope glycoprotein to generate an enhanced immune response as compared to wildtype; said method comprising the steps of:

- Selecting at least one HIV envelope glycoprotein among the group consisting of GP41 and GP160;
- Modifying the immunosuppressive domain of said envelope glycoprotein, said immunosuppressive domain being localizable by the sequence:

L
₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀X₁₁X₁₂X₁₃X₁₄X₁₅X₁₆X₁₇X₁₈X₁₉X₂₀X₂₁G₂₂C₂₃X₂₄

X₂₅X₂₆X₂₇X₂₈C₂₉

- Where X is any amino acid residue;
- Said modifying step comprising mutating or genetically modifying at least one of the amino acids selected among the amino acids X₂-X₁₇, whereby said sequence allows maintaining the ability of expression of said HIV-1 envelope glycoprotein on the surface of cells or virus, and further provides reduced immune suppression of said HIV-1 envelope glycoprotein, and wherein said amino acids after said modifying step are selected among:

X2: Q, R, N, A, G

X3: A, T, V, G, Q, R

X4: R, K, G, A, Q

X5: V, I, L, M, A, G, Q, R

X6: L, A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, Y

X7: A, T, S, G, Q, R

X8: V, I, L, Q, M, A, G, Q, R

X9: E, K, A, G, Q, R

X10: R, S, T, K, G, N, E, D, A, G, Q

X11: L, A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, Y

X12: L, V, I, M, R, A, G, Q

X13: K, Q, R, A, E, G, S, H, W, T, V

X14: D, E, N, H, G, A, G, Q, R

X15: Q, R, A, G

X16: Q, A, S, K, T, R, H, G

X17: L, M, S, Q, R, H, I, V, F, A, G

Said modifying step is not necessarily limited to modifying at least one of the amino acids selected among the amino acids X₂-X₁₇. As an example, even one, two, three or all of the amino acids L₁, G₂₂, C₂₃and/or C₂₉may be modified, as long as the obtained sequence allows maintaining the ability of expression of said HIV-1 envelope glycoprotein on the surface of cells or virus, and further provides reduced immune suppression of the obtained HIV-1 envelope glycoprotein. It is preferred that the amino acids L₁, G₂₂, C₂₃and C₂₉are not modified.

The term “localizable” is used to express that the relevant domain can be localized using the indicated sequence.

According to another aspect, the invention concerns an HIV-1 envelope glycoprotein obtainable according to the invention

According to an aspect, the invention concerns an antigen obtainable by selecting a part of an HIV-1 envelope glycoprotein, which HIV-1 envelope glycoprotein is obtainable according to the invention, said part comprising the modified domain of said envelope glycoprotein.

The expression “a part” in this context refers to any part.

According to an aspect, the invention concerns a nucleic acid sequence, preferably recombinant, encoding an HIV-1 envelope glycoprotein, an HIV-1 envelope polypeptide or an antigen according to the invention.

According to an aspect, the invention concerns an isolated eukaryotic expression vector comprising a nucleic acid sequence according to the invention

According to an aspect, the invention concerns a vaccine composition for HIV, comprising an HIV-1 envelope glycoprotein according to the invention.

According to an aspect, the invention concerns a method for producing an antibody, said method comprising the steps of:

- Administering an entity selected among an HIV-1 envelope glycoprotein, an HIV-1 envelope polypeptide, an antigen, a nucleic acid sequence or a vector according to the invention to a host, such as an animal; and
- Obtaining the antibody from said host.

According to an aspect, the invention concerns an antibody obtainable according to invention.

According to an aspect, the invention concerns neutralizing antibodies obtained or identified by the use of at least one HIV-1 envelope glycoprotein according to the invention.

According to an aspect, the invention concerns a method for manufacturing neutralizing antibodies comprising the use of at least one HIV-1 glycoprotein according to the invention.

According to an aspect, the invention concerns a method for manufacturing humanized neutralizing antibodies, comprising the use of at least one sequence selected among the sequences 1 to 336.

According to an aspect, the invention concerns a vaccine composition, comprising an entity selected among the group consisting of an HIV-1 envelope glycoprotein, an HIV-1 envelope polypeptide, an antigen, a nucleic acid sequence, a vector and an antibody according to the invention, and in addition at least one excipient, carrier or diluents.

According to an aspect, the invention concerns a method for producing a vaccine composition according to the invention comprising combining at least one HIV-1 envelope glycoprotein, an HIV-1 envelope polypeptide, an antigen, a nucleic acid sequence, a vector or an antibody according to the invention, preferably in purified form, with at least one adjuvant

According to an aspect, the invention concerns a pharmaceutical composition comprising an HIV-1 envelope glycoprotein, an HIV-1 envelope polypeptide, an antigen, a nucleic acid sequence, a vector, an antibody or a vaccine composition according to the invention, and at least one pharmaceutically acceptable excipient.

According to an aspect, the invention concerns a use of an HIV-1 envelope glycoprotein or an HIV-1 envelope polypeptide, or a dimerized version or a trimerized version or a multimerized version of the purified envelope polypeptide according to the invention as an antigen.

According to an aspect, the invention concerns a use of an HIV-1 envelope glycoprotein, an HIV-1 envelope polypeptide, an antigen, a nucleic acid sequence, a vector or an antibody according to the invention, for a medical purpose, such as for the treatment, amelioration or prevention of a clinical condition, such as for the manufacture of a medicament for the treatment, amelioration or prevention of a clinical condition, such as HIV.

According to an aspect, the invention concerns a method of treating or ameliorating the symptoms of an individual infected with HIV, or prophylactic treating an individual against HIV infection, comprising administering an amount of HIV-1 envelope glycoprotein, HIV-1 envelope polypeptide, antigen, nucleic acid sequence, vector or vaccine composition according to the invention.

In one aspect, the present invention relates to an HIV-1 envelope polypeptide which encompasses a region of 29 amino acids further comprising the mutated immunosuppressive domain rendering said immunosuppressive domain non-immunosuppressive where the amino acids at position 1, 22, 23 and 29 are invariable with the amino acid at position 1 being a Leucine, the amino acid at position 22 a Glycine, the amino acids at position 23 a Cysteine and the amino acid at position 29 a Cysteine. As such the HIV envelope antigen for making an HIV vaccine or generating neutralizing antibodies against HIV should contain the following sequence

<seqid 1;PRT;>:

L
₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀X₁₁X₁₂X₁₃X₁₄X₁₅X₁₆X₁₇X₁₈X₁₉X₂₀X₂₁G₂₂C₂₃X₂₄

X₂₅X₂₆X₂₇X₂₈C₂₉

Where X is any amino acid residue.

DETAILED DISCLOSURE

In a preferred embodiment the antigen should encompass a sequence where the amino acids has been even more restricted to

<seqid 2;PRT;>:

L
₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀X₁₁X₁₂X₁₃X₁₄X₁₅X₁₆X₁₇X₁₈X₁₉X₂₀X₂₁G₂₂C₂₃X₂₄X₂₅X₂₆X₂₇X₂₈C₂₉

where

X is

X1: L

X2: Q, R, N, A, G

X3: A, T, V, G, Q, R

X4: R, K, G, A, Q

X5: V, I, L, M, A, G, Q, R

X6: L, A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, Y

X7: A, T, S, G, Q, R

X8: V, I, L, Q, M, A, G, Q, R

X9: E, K, A, G, Q, R

X10: R, S, T, K, G, N, E, D, A, G, Q

X11: L, A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, Y

X12: L, V, I, M, R, A, G, Q

X13: K, Q, R, A, E, G, S, H, W, T, V

X14: D, E, N, H, G, A, G, Q, R

X15: Q, R, A, G

X16: Q, A, S, K, T, R, H, G

X17: L, M, S, Q, R, H, I, V, F, A

X18: L, M

X19: G, N, S, A, Q, R

X20: I, L, S, A, T, E, V, M, F

X21: W, F

X22: G

X23: C

X24: S, K, T, Q, R, A

X25: G, F, D, N, W

X26: K, N, R, F, M, Q, A, S

X27: L, H, P, A, Q, S, T, W, I, V, F, R

X28: I, V, T

X29: C

In yet a preferred embodiment the antigen should encompass a sequence where the amino acids has been even further restricted to

<seq id 3; PRT;>:

L
₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀X₁₁X₁₂X₁₃X₁₄X₁₅X₁₆X₁₇X₁₈X₁₉X₂₀X₂₁G₂₂C₂₃X₂₄X₂₅X₂₆X₂₇X₂₈C₂₉

where

X is

X1: L

X2: Q, R, A, G, Q

X3: A, T, G, Q, R

X4: R, K, G, A, G, Q

X5: V, I, L, A, G, Q, R

X6: L, A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, Y

X7: A, S, T, G, Q, R

X8: V, I, L, M, A, G, Q, R

X9: E, K, A, G, Q, R

X10: R, S, T, K, G, A, Q

X11: L, A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, Y

X12: L, V, I, M, A, G, Q, R

X13: K, Q, R, A, E, G, S, H, W, T, V, G

X14: D, N, G, A, Q, R

X15: Q, R, A, G

X16: Q, K, T, R, H, A, G

X17: L, R, I, F, G, Q

X18: L, M

X19: G, N, S, R

X20: I, L, S, A, T, E, V, M, F

X21: W

X22: G

X23: C

X24: S, K, T, R, A

X25: G

X26: K, N, R

X27: L, H, P, A, Q, S, T, W, I, V, F, R

X28: I, V

X29: C

In an even more preferred embodiment the antigen should encompass a sequence where the amino acids has even been further restricted to

<seqid 4;PRT;>:

L
₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀X₁₁X₁₂X₁₃X₁₄X₁₅X₁₆X₁₇X₁₈X₁₉X₂₀X₂₁G₂₂C₂₃X₂₄X₂₅X₂₆X₂₇X₂₈C₂₉

where

X is

X1: L

X2: Q, R, A, G

X3: A, T, G, Q, R

X4: R, K, A, G, Q

X5: V, I, L, A, G, Q, R

X6: L, A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, Y

X7: A, S, T, A, G, Q, R

X8: V, I, L, A, G, Q, R

X9: E, K, A, G, Q, R

X10: R, S, T, K, G, A, Q

X11: L, A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, Y

X12: L, V, I, M, A, G, Q, R

X13: K, Q, R, A, E, G, S, H, W, T, V, A, G

X14: D, N, A, G, Q, R

X15: Q, R, A, G

X16: Q, K, T, R, A, G

X17: L, R, I, F, A, G, Q

X18: L

X19: G, N, S, R

X20: I, L, S, V, F

X21: W

X22: G

X23: C

X24: S, K, T, A

X25: G

X26: K, R

X27: L, H, P, A, Q, S, T, W, I, V, F, R

X28: I, V

X29: C

Particularly preferred sequences are sequences selected among sequences with seqID 5 to seqID 228.

Examples of HIV envelopes suited as antigens for HIV vaccination or for generating neutralizing antibodies against HIV are given but not restricted to seqid 229-366. These sequences represent different HIV clades or combinations thereof, which an HIV vaccine must be effective against.

Another aspect relates to a method of treating, preventing or ameliorating a clinical condition, said method comprising administering to an individual suffering from said clinical condition an effective amount of an HIV-1 envelope polypeptide or part thereof as defined in the present invention, an antigen as defined in the present invention, a nucleic acid as defined in the present invention, a vector as defined in the present invention, a biological entity as defined in the present invention, a vaccine composition as defined in the present invention, or a kit-of-parts as defined in the present invention.

Terms

HIV-1 envelope proteins are produced as a polyprotein (gp160) which is cleaved into its two subunits gp120 and gp41. Examples of such HIV envelopes is exemplified by but not restricted to SEQ ID NO: 229-366. Mutations can be introduced into the HIV envelope polyprotein (gp160) rendering it resistant to cleavage into its two subunits (gp120 and gp41). Such HIV envelope mutants are considered bona fide envelope antigens suited for introduction of further mutations rendering them non-immunosuppressive.

HIV: As used herein, the term “HIV” refers to all forms, subtypes and variations of the HIV virus, and is synonymous with the older terms for HIV, such as HTLV III and LAV. Various cell lines capable of propagating HIV or permanently infected with the HIV virus have been developed and deposited with the ATCC, including HuT 78 cells and the HuT 78 derivative H9, as well as those having accession numbers CCl 214, TIB 161, CRL 1552 and CRL 8543, which are described in U.S. Pat. No. 4,725,669 and Gallo, Scientific 3 0 American 256:46 (1987).

Vaccination modality: A vaccination modality is defined as any step or usage of the immunogen described in this application for any vaccination purpose or part thereof.

Partial knock out of the immunosuppressive domain: Is defined as at least a 30% reduction of the immunosuppression elicited in the CTLL-2 assay by an dimerized mutant peptide as defined in SEQid 1 as compared to a wildtype dimerized peptide as defined in Segid 369.

According to an embodiment, the inventions concerns a method for making an HIV-1 envelope glycoprotein, which may be expressed on the surface of cells, virus or virus like particles, and which provides reduced immune suppression, allowing said HIV-1 envelope glycoprotein to generate an enhanced immune response as compared to wildtype; said method comprising the steps of:

- Selecting at least one HIV envelope glycoprotein among the group consisting of GP41 and GP160;
- Modifying the immunosuppressive domain of said envelope glycoprotein, said immunosuppressive domain being localizable by the sequence:

L
₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀X₁₁X₁₂X₁₃X₁₄X₁₅X₁₆X₁₇X₁₈X₁₉X₂₀X₂₁G₂₂C₂₃X₂₄X₂₅X₂₆X₂₇X₂₈C₂₉

- Where X is any amino acid residue;
- Said modifying step comprising mutating or genetically modifying at least one of the amino acids selected among the amino acids X₂-X₁₇, whereby said sequence allows maintaining the ability of expression of said HIV-1 envelope glycoprotein on the surface of cells or virus, and further provides reduced immune suppression of said HIV-1 envelope glycoprotein, and wherein said amino acids after said modifying step are selected among:

According to an embodiment, the inventions concerns a method for making an HIV-1 envelope glycoprotein, which may be expressed on the surface of cells or virus, and which provides reduced immune suppression, allowing said HIV-1 envelope glycoprotein to generate an enhanced immune response as compared to wildtype; said method comprising the steps of:

- Selecting at least one HIV envelope glycoprotein among the group consisting of GP41 and GP160;
- Modifying the immunosuppressive domain of said envelope glycoprotein, said immunosuppressive domain being localizable by the sequence:

L
₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀X₁₁X₁₂X₁₃X₁₄X₁₅X₁₆X₁₇X₁₈X₁₉X₂₀X₂₁G₂₂C₂₃X₂₄X₂₅X₂₆X₂₇X₂₈C₂₉

- Said modifying step comprising mutating or genetically modifying at least one of the amino acids selected among the amino acids X₂-X₁₇into any amino acid, whereby said sequence allow maintaining the ability to expression on the surface of cells or virus, and further provides reduced immune suppression of said HIV-1 envelope glycoprotein.

According to an embodiment, the inventions concerns a method according to the invention, for making an HIV-1 envelope glycoprotein for medical use, such as therapeutic or prophylactic purpose, preferably for use as a vaccine.

According to an embodiment, the invention concerns a method, for making an HIV-1 envelope glycoprotein for vaccination purposes or for the generation of neutralizing antibodies.

According to an embodiment, the invention concerns a method, wherein said modifying step does not provide a sequence selected among a wild type sequence having immune suppressing properties.

According to an embodiment, the invention concerns a method, wherein said modifying step does not provide a sequence selected among the sequences:

X2: Q, R

X3: A, T

X4: R

X5: V, I, L

X6: L

X7: A

X8: V, I, L, M

X9: E

X10: R, S, T, K, G

X11: Y, L, F

X12: L, I, V

X13: K, Q, R, G, S

X14: D, N

X15: Q

X16: Q, K, R

X17: L, I, F

According to an embodiment, the invention concerns a method, wherein said modifying step does not provide a sequence selected among the sequences:

X2: Q, R

X3: A, T

X4: R

X5: V, I, L

X6: L

X7: A

X8: V, I, L, M

X9: E

X10: R, S, T, K

X11: Y, L, F

X12: L, I

X13: K, Q, R, G

X14: D, N

X15: Q

X16: Q, K, R

X17: L, F

According to an embodiment, the invention concerns a method, wherein the HIV-1 envelope glycoprotein may be expressed on the surface of virus, as said sequence provides the ability of expression on the virus to said HIV-1 envelope glycoprotein.

According to an embodiment, the invention concerns a method, wherein the HIV-1 envelope glycoprotein exhibits fusiogenic activity, as said sequence further provides fusiogenic activity to said HIV-1 envelope glycoprotein.

The method according to any of the previous claims, wherein said modifying step provides a sequence selected among:

The method according to claim 10, wherein said modifying step provides a sequence selected among:

X2: Q, R, A, G,

X3: A, T, G, Q, R

X4: R, K, G, A, G, Q

X5: V, I, L, A, G, Q, R

X6: L, A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, Y

X7: A, S, T, G, Q, R

X8: V, I, L, M, A, G, Q, R

X9: E, K, A, G, Q, R

X10: R, S, T, K, G, A, Q

X11: L, A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, Y

X12: L, V, I, M, A, G, Q, R

X13: K, Q, R, A, E, G, S, H, W, T, V, G

X14: D, N, G, A, Q, R

X15: Q, R, A, G

X16: Q, K, T, R, H, A, G

X17: L, R, I, F, G, Q, A

The method according to claim 11, wherein said modifying step provides a sequence selected among:

X2: Q, R, A, G

X3: A, T, G, Q, R

X4: R, K, A, G, Q

X5: V, I, L, A, G, Q, R

X6: L, A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, Y

X7: A, S, T, A, G, Q, R

X8: V, I, L, A, G, Q, R

X9: E, K, A, G, Q, R

X10: R, S, T, K, G, A, Q

X11: L, A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, Y

X12: L, V, I, M, A, G, Q, R

X13: K, Q, R, A, E, G, S, H, W, T, V, A, G

X14: D,N, A, G, Q, R

X15: Q, R, A, G

X16: Q, K, T, R, A, G

X17: L, R, I, F, A, G, Q