WO2019069273A1

WO2019069273A1 - Recombinant mva with modified hiv-1 env

Info

Publication number: WO2019069273A1
Application number: PCT/IB2018/057731
Authority: WO
Inventors: Anna-Lise Williamson; Edward Peter Rybicki; Michiel VAN DIEPEN; Nicola Jennifer DOUGLASS; Rosamund Eira CHAPMAN
Original assignee: University Of Cape Town
Priority date: 2017-10-04
Filing date: 2018-10-04
Publication date: 2019-04-11
Also published as: CN111417405A; GB201716181D0

Abstract

The present invention relates to a prime-boost vaccine comprising a priming HIV-1 immunogen and one or more boosting HIV-1 immunogens, wherein the priming HIV- 1 immunogen comprises or consists of either (a) a DNA expression vector capable of expressing a modified HIV-1 gp150 envelope protein in a mammalian cell, or (b) a recombinant modified vaccinia Ankara (MVA) virus, including a nucleic acid encoding a modified HIV-1 gp150 envelope protein; and wherein the one or more boosting HIV-1 immunogens comprises: (a) a recombinant modified vaccinia Ankara (MVA) virus, including a nucleic acid encoding a modified HIV-1 gp150 envelope protein; and/or (b) a soluble modified HIV-1 gp140 envelope protein. Further the invention also relates to the prime-boost vaccine for use in a method of inducing an immune response, methods of inducing an immune response to HIV-1 in a subject and to kits including the priming composition and boosting compositions of the invention.

Description

RECOMBINANT MVA WITH MODIFIED HIV-1 ENV

BACKGROUND OF THE INVENTION

The present invention relates to a prime-boost vaccine comprising a priming HIV-1 immunogen and one or more boosting HIV-1 immunogens, wherein the priming HIV-1 immunogen comprises or consists of either (a) a DNA expression vector capable of expressing a modified HIV-1 gp150 envelope protein in a mammalian cell, or (b) a recombinant modified vaccinia Ankara (MVA) virus, including a nucleic acid encoding a modified HIV-1 gp150 envelope protein; and wherein the one or more boosting HIV-1 immunogens comprises: (a) a recombinant modified vaccinia Ankara (MVA) virus, including a nucleic acid encoding a modified HIV-1 gp150 envelope protein; and/or (b) a soluble modified HIV-1 gp140 envelope protein. Further the invention also relates to the prime-boost vaccine for use in a method of inducing an immune response to HIV-1 in a subject comprising the aforementioned priming and boosting HIV-1 immunogens. The invention also includes methods of inducing an immune response to HIV-1 in a subject comprising the administration of the priming and boosting HIV-1 immunogens and to kits including the priming composition and boosting compositions of the invention.

The HIV pandemic is a global public health challenge and is particularly problematic in developing countries, which are often disproportionately affected and lack the infrastructure necessary for manufacturing of their own vaccines. A major focal point of current HIV vaccine research is the development of vaccines capable of inducing neutralizing antibodies. The type of neutralizing antibody is important and there is a hierarchy of viruses according to how difficult they are to neutralize, with Tier 1 a viruses being easiest to neutralise and Tier 2 being more resistant to neutralisation. Convincing cross-neutralizing antibodies against tier 2 viruses have not been elicited by vaccination in either animal models or humans, and only a few studies have reported the induction of neutralizing antibodies to the vaccine matched strain (i.e. autologous neutralizing antibodies) in preclinical animal models. In many cases these autologous neutralizing antibodies were reported at low titres or are only inconsistently elicited in a subset of animals. A major focal point of current HIV vaccine research is the development of native-like envelope trimers that are capable of inducing neutralizing antibodies. These antigens are typically produced by transient transfection of mammalian cells, such as HEK 293T, HEK 293F, CHO-K1 or GnTI-/- cell lines. The only effective HIV vaccine efficacy trial to date included a poxvirus prime and a boost with protein. This trial (RV144) used the avipoxvirus, ALVAC. Tier two neutralising antibodies were not induced in this trial.

Only a few groups have been able to successfully elicit Tier 2 neutralizing antibodies using a prime-boost immunization strategy:

Townsley et al. (2016) described utilizing a replicating vaccinia virus as a vector and expressed gp120 envelope protein, whereas the present inventors have produced a modified vaccinia Ankara (MVA) expressing trimeric, gp150 envelope protein including a flexible linker in place of the furin cleavage site.

Cappucci et al. (2017) describe the use of MVA to produce neutralizing antibodies. The MVA used by these authors expresses a soluble, cleaved gp140 envelope protein and not a membrane-bound gp150 protein as described by the present inventors.

SUMMARY OF THE INVENTION

In a first aspect of the invention there is provided for a prime-boost vaccine comprising a priming HIV-1 immunogen and one or more boosting HIV-1 immunogens, wherein the priming HIV-1 immunogen comprises either (a) a DNA expression vector capable of expressing a modified HIV-1 gp150 envelope protein in a mammalian cell; or (b) a recombinant modified vaccinia Ankara (MVA) virus, including a nucleic acid encoding a modified HIV-1 gp150 envelope protein, and wherein the one or more boosting HIV-1 immunogens comprises (a) a recombinant modified vaccinia Ankara (MVA) virus, including a nucleic acid encoding a modified HIV-1 gp150 envelope protein; and/or (b) a soluble modified HIV-1 gp140 envelope protein.

In one embodiment of the invention the modified HIV-1 gp150 envelope protein comprises a truncated cytoplasmic domain, a flexible glycine linker replacing the furin cleavage site and an I559P mutation. Additionally, the modified HIV-1 gp140 envelope protein comprises a flexible glycine linker replacing the furin cleavage site and an I559P mutation.

In another embodiment of the invention the MVA or DNA expression vector optionally includes a nucleic acid encoding an HIV-1 Gag protein. Preferably the HIV- 1 Gag protein is substantially identical to the sequence of SEQ ID NO:1 1 .

In a further embodiment of the invention the modified HIV-1 gp150 envelope protein is substantially identical to the sequence of SEQ ID NO:3 or SEQ ID NO:15, the modified HIV-1 gp140 envelope protein is substantially identical to the sequence of SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:17 or SEQ ID NO:21 . A further embodiment of the invention provides for a modified HIV-1 gp120 envelope protein substantially identical to the sequence of SEQ ID NO:7 or SEQ ID NO:19.

In yet a further embodiment of the invention the nucleic acid encoding the modified HIV-1 gp150 envelope protein includes a tissue plasminogen activator leader sequence.

In a preferred embodiment of this aspect the priming HIV-1 immunogen is the DNA expression vector capable of expressing a modified HIV-1 gp150 envelope protein in the mammalian cell, a first boosting HIV-1 immunogen comprises the recombinant modified vaccinia Ankara (MVA) virus, including a nucleic acid encoding a modified HIV-1 gp150 envelope protein, and the second boosting HIV-1 immunogen comprises the soluble modified HIV-1 gp140 envelope protein. In this embodiment one or more consecutive doses of the priming HIV-1 immunogen and each boosting HIV-1 immunogen are administered in a treatment regime of DNA- DNA-MVA-MVA-Protein-Protein. It will however be appreciated that the treatment regime could be varied and could comprise DNA-DNA-Protein; DNA-MVA-Protein; DNA-MVA-MVA-Protein-Protein; DNA-DNA-MVA-Protein-Protein; DNA-DNA-MVA- MVA-Protein; DNA-DNA-Protein-Protein-Protein or MVA-MVA-Protein-Protein- Protein.

It will be appreciated that the priming HIV-1 immunogen may be administered in one, two or three consecutive doses. Further, the boosting HIV-1 immunogen may be administered to the subject in one or more doses after the priming inoculation. The boosting HIV-1 immunogen may be administered as a composition and may include at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine or at least ten subsequent inoculations with the boosting composition comprising an HIV-1 immunogen selected from the group consisting of the recombinant MVA and/or soluble modified HIV-1 env protein. Those of skill in the art will appreciate that each boosting inoculation may include a different HIV-1 immunogen.

It will be appreciated that the modified HIV-1 envelope protein is capable of folding into a trimeric conformation.

In a second aspect of the invention there is provided for a prime-boost vaccine for use in a method of inducing an immune response to HIV-1 in a subject, the method comprising administering one or more consecutive doses of a priming HIV-1 immunogen and one or more consecutive doses of a boosting HIV-1 immunogen to the subject, wherein the priming HIV-1 immunogen comprises either (a) a DNA expression vector capable of expressing a modified HIV-1 gp150 envelope protein in the subject, or (b) a recombinant modified vaccinia Ankara (MVA) virus, including a nucleic acid encoding a modified HIV-1 gp150 envelope protein; and wherein the boosting HIV-1 immunogen comprises (a) a recombinant modified vaccinia Ankara (MVA) virus, including a nucleic acid encoding a modified HIV-1 gp150 envelope protein; and/or (b) a soluble modified HIV-1 gp140 envelope protein.

In another embodiment of the invention the MVA or DNA expression vector optionally includes a nucleic acid encoding an HIV-1 Gag protein. Preferably, the HIV-1 Gag protein is substantially identical to the sequence of SEQ ID NO:1 1 .

In a preferred embodiment of this aspect of the invention the priming HIV-1 immunogen is the DNA expression vector capable of expressing a modified HIV-1 gp150 envelope protein in the subject and is administered in one or more consecutive doses. The first boosting HIV-1 immunogen comprises the recombinant modified vaccinia Ankara (MVA) virus, including a nucleic acid encoding a modified HIV-1 gp150 envelope protein and is administered in one or more consecutive doses, and the second boosting HIV-1 immunogen comprises the soluble modified HIV-1 gp140 envelope protein and is administered in one or more consecutive doses. In this embodiment of the invention the administration regime is DNA-DNA-MVA-MVA- Protein-Protein.

In a further embodiment the immune response is a neutralising antibody response or a cytotoxic T lymphocyte response.

In still a further embodiment the subject is a human.

In a third aspect of the invention there is provided for a method of inducing an immune response to HIV-1 in a subject, the method comprising administering one or more consecutive doses of a priming HIV-1 immunogen and one or more consecutive doses of a boosting HIV-1 immunogen to the subject, wherein the priming HIV-1 immunogen comprises either (a) a DNA expression vector capable of expressing a modified HIV-1 gp150 envelope protein in the subject; or (b) a recombinant modified vaccinia Ankara (MVA) virus, including a nucleic acid encoding a modified HIV-1 gp150 envelope protein; and wherein the boosting HIV-1 immunogen comprises (a) a recombinant modified vaccinia Ankara (MVA) virus, including a nucleic acid encoding a modified HIV-1 gp150 envelope protein; and/or (b) a soluble modified HIV-1 gp140 envelope protein.

In another embodiment of the invention the MVA or DNA expression vector optionally includes a nucleic acid encoding an HIV-1 Gag protein.

In a further embodiment of the invention the modified HIV-1 gp150 envelope protein is substantially identical to the sequence of SEQ ID NO:3 or SEQ ID NO:15, the modified the modified HIV-1 gp140 envelope protein is substantially identical to the sequence of SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:17 or SEQ ID NO:21 . Additionally, there is provided for a modified HIV-1 gp120 envelope protein substantially identical to the sequence of SEQ ID NO:7 or SEQ ID NO:19.

In a preferred embodiment of this aspect of the invention wherein the priming HIV-1 immunogen is the DNA expression vector capable of expressing a modified HIV-1 gp150 envelope protein in the subject and is administered in one or more consecutive doses. The first boosting HIV-1 immunogen comprises the recombinant modified vaccinia Ankara (MVA) virus, including a nucleic acid encoding a modified HIV-1 gp150 envelope protein and is administered in one or more consecutive doses after the priming immunogen, and the second boosting HIV-1 immunogen comprises the soluble modified HIV-1 gp140 envelope protein and is administered to the subject in one or more consecutive doses after the first boosting immunogen In this embodiment of the invention the administration regime is DNA-DNA-MVA-MVA- Protein-Protein

In still a further embodiment the subject is a human.

In a fourth aspect of the invention there is provided for a kit for inducing an immune response to HIV-1 infection in a subject comprising a priming composition, and at least one boosting composition, wherein the priming composition comprises an HIV-1 immunogen selected from either (a) a DNA expression vector capable of expressing a modified HIV-1 gp150 envelope protein in the subject; or (b) a recombinant modified vaccinia Ankara (MVA) virus, including a nucleic acid encoding a modified HIV-1 gp150 envelope protein; and wherein the at least one boosting composition comprises an HIV-1 immunogen selected from (a) a recombinant modified vaccinia Ankara (MVA) virus, including a nucleic acid encoding a modified HIV-1 gp150 envelope protein; and/or (b) a soluble modified HIV-1 gp140 envelope protein, and instructions for use to administer one or more consecutive doses of each priming composition and each boosting HIV-1 composition the subject.

The present invention also relates to a method for eliciting an immune response to HIV-1 in a subject, the method comprising, administering a prime-boost vaccine comprising a priming composition and a boosting composition to the subject, wherein the priming composition comprises an HIV-1 antigen selected from either: (a) a DNA expression vector capable of expressing a modified HIV-1 gp150 envelope protein; or (b) a recombinant modified vaccinia Ankara (MVA) virus, including a nucleic acid encoding a modified HIV-1 gp150 envelope protein; and wherein the boosting composition comprises at least two HIV-1 antigens selected from (a) to (c). Further, the invention also relates to a prime-boost vaccine comprising the aforementioned priming composition and boosting compositions. The invention also includes the use of the vaccine in a method of eliciting an immune response and a kit including the priming composition and boosting compositions of the invention.

In a fifth aspect of the invention there is provided for a method for eliciting an immune response to HIV-1 in a subject, the method comprising the steps of administering a prime-boost vaccine to the subject, wherein the prime-boost vaccine comprises a priming composition and a boosting composition, the priming composition of the invention comprises an HIV-1 antigen selected from the group consisting of (a) a DNA expression vector capable of expressing a modified HIV-1 gp150 envelope protein in the subject, (b) a recombinant modified vaccinia Ankara (MVA) virus, including a nucleic acid encoding a modified HIV-1 gp150 envelope protein; or (c) a soluble modified HIV-1 gp140 envelope protein; the boosting composition of the invention comprises at least two independent administrations of an HIV-1 antigen, wherein the HIV-1 antigens in the boosting composition are selected from at least two different HIV-1 antigens in the group consisting of (a) a DNA expression vector capable of expressing a modified HIV-1 gp150 envelope protein in the subject, (b) a recombinant modified vaccinia Ankara (MVA) virus, including a nucleic acid encoding a modified HIV-1 gp150 envelope protein; or (c) a soluble modified HIV-1 gp140 envelope protein. In one embodiment of the invention the MVA or DNA expression vector optionally includes a nucleic acid encoding an HIV-1 Gag protein.

In another embodiment of the invention the modified HIV-1 gp150 envelope protein is substantially identical to the sequence of SEQ ID NO:3 or SEQ ID NO:15 and the modified HIV-1 gp140 envelope protein is substantially identical to the sequence of SEQ ID NO:5 or SEQ ID NO:17.

In a further embodiment of the invention the nucleic acid encoding the modified HIV-1 gp150 envelope protein includes a tissue plasminogen activator leader sequence.

It will be appreciated that any one of the HIV-1 antigens may be administered as the prime, the boost or both.

In a preferred embodiment of the invention the priming composition is the DNA expression vector capable of expressing a modified HIV-1 gp150 envelope protein in the subject; and the first boosting composition comprises the recombinant modified vaccinia Ankara (MVA) virus, including a nucleic acid encoding a modified HIV-1 gp150 envelope protein, and the second boosting composition comprises the soluble modified HIV-1 gp140 envelope protein.

It will be appreciated that the boost composition may include HIV-1 antigens which are administered to the subject in two or more doses after the initial priming inoculation. The boosting composition may include at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine or at least ten subsequent inoculations with a boosting composition comprising an HIV-1 antigen selected from the group consisting of the recombinant MVA, the DNA expression vector encoding a modified HIV-1 env and/or soluble modified HIV-1 env protein. Those of skill in the art will appreciate that each boosting inoculation may include a different HIV-1 antigen.

In yet a further embodiment the immune response is a neutralising antibody response or a cytotoxic T lymphocyte response.

Preferably, the modified HIV-1 envelope protein that is expressed on the MVA, from the expression vector or is in a soluble protein form is capable of folding into a trimeric conformation.

In a preferable embodiment of the invention the subject is a human.

In a sixth aspect of the invention there is provided for a prime-boost vaccine comprising a priming composition and a boosting composition, wherein the priming composition comprises an HIV-1 antigen selected from the group consisting of: (a) a DNA expression vector capable of expressing a modified HIV-1 gp150 envelope protein in a mammalian cell, (b) a recombinant modified vaccinia Ankara (MVA) virus, including a nucleic acid encoding a modified HIV-1 gp150 envelope protein; or (c) a soluble modified HIV-1 gp140 envelope protein; and wherein the boosting composition comprises at least two HIV-1 antigens selected from the group consisting of (a) a DNA expression vector capable of expressing a modified HIV-1 gp150 envelope protein in the subject, (b) a recombinant modified vaccinia Ankara (MVA) virus, including a nucleic acid encoding a modified HIV-1 gp150 envelope protein; or (c) a soluble modified HIV-1 gp140 envelope protein.

In one embodiment of the invention the MVA or DNA expression vector optionally includes a nucleic acid encoding an HIV-1 Gag protein.

In another embodiment of the invention the modified HIV-1 gp150 envelope protein is substantially identical to the sequence of SEQ ID NO:3 or SEQ ID NO:15 and the the modified HIV-1 gp140 envelope protein is substantially identical to the sequence of SEQ ID NO:5 or SEQ ID NO:17.

It will be appreciated that the prime composition, the boost composition or both may comprises any one of the HIV-1 antigens.

In a preferred embodiment of the invention the priming composition is the DNA expression vector capable of expressing a modified HIV-1 gp150 envelope protein in the mammalian cell; and wherein a first boosting composition comprises the recombinant modified vaccinia Ankara (MVA) virus, including a nucleic acid encoding a modified HIV-1 gp150 envelope protein, and wherein a second boosting composition comprises the soluble modified HIV-1 gp140 envelope protein.

In a seventh aspect of the invention there is provided for a prime-boost vaccine comprising a priming composition and a boosting composition, wherein the priming composition comprises an HIV-1 antigen selected from the group consisting of (a) a DNA expression vector capable of expressing a modified HIV-1 gp150 envelope protein in the subject, (b) a recombinant modified vaccinia Ankara (MVA) virus, including a nucleic acid encoding a modified HIV-1 gp150 envelope protein; or (c) a soluble modified HIV-1 gp140 envelope protein; and wherein the boosting composition comprises at least two HIV-1 antigens selected from the group consisting of (a) a DNA expression vector capable of expressing a modified HIV-1 gp150 envelope protein in the subject, (b) a recombinant modified vaccinia Ankara (MVA) virus, including a nucleic acid encoding a modified HIV-1 gp150 envelope protein; or (c) a soluble modified HIV-1 gp140 envelope protein; for use in a method of eliciting an immune response to HIV-1 in a subject, the method comprising, administering the prime-boost vaccine to the subject.

In a preferred embodiment of the invention the priming composition is the DNA expression vector capable of expressing a modified HIV-1 gp150 envelope protein in the subject, and wherein a first boosting composition comprises the recombinant modified vaccinia Ankara (MVA) virus, including a nucleic acid encoding a modified HIV-1 gp150 envelope protein, and wherein a second boosting composition comprises the soluble modified HIV-1 gp140 envelope protein.

In a preferable embodiment of the invention the subject is a human.

In an eighth aspect of the invention there is provided for a kit for eliciting an immune response to HIV-1 infection in a subject. Wherein the kit comprises a first container and at least two further containers. The first container comprises a priming composition, and wherein the at least two further containers each contain a boosting composition, wherein the priming composition comprises an HIV-1 antigen selected from the group consisting of (a) a DNA expression vector capable of expressing a modified HIV-1 gp150 envelope protein in the subject, (b) a recombinant modified vaccinia Ankara (MVA) virus, including a nucleic acid encoding a modified HIV-1 gp150 envelope protein; or (c) a soluble modified HIV-1 gp140 envelope protein; and wherein each boosting composition comprises a different HIV-1 antigen selected from the group consisting of (a) a DNA expression vector capable of expressing a modified HIV-1 gp150 envelope protein in the subject, (b) a recombinant modified vaccinia Ankara (MVA) virus, including a nucleic acid encoding a modified HIV-1 gp150 envelope protein, or (c) a soluble modified HIV-1 gp140 envelope protein.

BRIEF DESCRIPTION OF THE FIGURES

Non-limiting embodiments of the invention will now be described by way of example only and with reference to the following figures:

Figure 1 : Modifications made to the native gp160 HIV-1 envelope protein. In addition the tissue plasminogen activator (tPA) leader sequence was added to the amino terminus of the protein sequence for expression in MVA. The gp150, gp120HA2 and gp140HA2tr were expressed in MVA.

Figure 2: Transfer vector for construction of recombinant MVA. G1 L flank = portion of the G1 L gene. GP41 = gp41 region of envelope gene. GP120-Gly linker = gp120 portion of the envelope gene + (GGGGSGGGGS), tPA = tissue plasminogen activator leader sequence, mH5 promoter = poxvirus mH5 promoter, K1 L = K1 L host range selection gene, pSS promoter = pSS poxvirus promoter, eGFP = green fluorescent protein gene, p7.5 promoter = p7.5 poxvirus promoter, I8R flank = portion of the I8R gene, ColE1 Origin = Escherichia coli plasmid ColE1 origin of replication, CmpR = chloramphenicol resistance gene.

Figure 3: Correlation of Tier 2 neutralization with lower binding antibody end point titres for MVAGC5 and MVAC5 primed animals (^*p<0.05, ^**p<0.01 ).

Figure 4: Serum binding antibodies elicited to HIV-1 Du151 Env following priming with MVAGD5 or SAAVI MVA-C.

Figure 5: A comparison of binding antibody titres to Env. Rabbits were vaccinated with either a DNA prime-protein boost, with and without Gag (DNAGC5 / DNAC5), a MVA prime-protein boost with and without Gag (MVAGC5 / MVAC5) or protein only. Figure 6: A comparison of Tier 2 antibody titres to Env. Rabbits were vaccinated with either a DNA prime-protein boost, with and without Gag (DNAGC5 / DNAC5), a MVA prime-protein boost, with and without Gag (MVAGC5 / MVAC5) or protein only.

SEQUENCE LISTING

The nucleic acid and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and the standard three letter abbreviations for amino acids. It will be understood by those of skill in the art that only one strand of each nucleic acid sequence is shown, but that the complementary strand is included by any reference to the displayed strand. In the accompanying sequence listing:

SEQ ID NO:1 - Amino acid sequence of the HIV-1 gp1 60 envelope protein from CAP256. SEQ ID NO:2 - Nucleic acid sequence encoding the HIV-1 gp160 envelope protein from

CAP256.

SEQ ID NO:3 - Amino acid sequence of the modified CAP256 gp150 polypeptide.

SEQ ID NO:4 - Nucleic acid sequence encoding the modified CAP256 gp150 polypeptide. SEQ ID NO:5 - Amino acid sequence of the modified CAP256 gp140 polypeptide.

SEQ ID NO:6 - Nucleic acid sequence encoding the modified CAP256 gp140 polypeptide SEQ ID NO:7 - Amino acid sequence of the modified CAP256 gp120HA2 polypeptide.

SEQ ID NO:8 - Nucleic acid sequence encoding the modified CAP256 gp120HA2

polypeptide.

SEQ ID NO:9 - Amino acid sequence of the modified CAP256 gp140HA2tr polypeptide SEQ ID NO:10 - Nucleic acid sequence encoding the modified CAP256 gp140HA2tr

polypeptide.

SEQ ID NO:1 1 - Amino acid sequence of the HIV-1 subtype C mosaic Gag protein.

SEQ ID NO:12 - Nucleic acid sequence encoding the HIV-1 subtype C mosaic Gag protein. SEQ ID NO:13 - Amino acid sequence of the HIV-1 gp160 envelope sequence from Du151 . SEQ ID NO:14 - Nucleic acid sequence of the HIV-1 gp160 envelope sequence from Du151 . SEQ ID NO:15 - Amino acid sequence of the modified Du151 gp150.

SEQ ID NO:16 - Nucleic acid sequence of the modified Du151 gp1 50.

SEQ ID NO:17 - Amino acid sequence of the modified Du151 gp140 polypeptide.

SEQ ID NO:18 - Nucleic acid sequence encoding the modified Du1 51 gp140 polypeptide. SEQ ID NO:19 - Amino acid sequence of the modified Du151 gp120HA2 polypeptide.

SEQ ID NO:20 - Nucleic acid sequence encoding the modified Du1 51 gp120HA2

polypeptide.

SEQ ID NO:21 - Amino acid sequence of the modified Du151 gp140HA2tr polypeptide. SEQ ID NO:22 - Nucleic acid sequence encoding the modified Du1 51 gp140HA2tr polypeptide.

SEQ ID NO:23 - Amino acid sequence of the Glycine-Serine based linker.

SEQ ID NO:24 - Nucleic acid sequence of the MVA transfer vector pSSPEx.

DETAILED DESCRIPTION OF THE INVENTION

The present invention will now be described more fully hereinafter with reference to the accompanying drawings, in which some, but not all embodiments of the invention are shown.

The invention as described should not be limited to the specific embodiments disclosed and modifications and other embodiments are intended to be included within the scope of the invention. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

As used throughout this specification and in the claims which follow, the singular forms "a", "an" and "the" include the plural form, unless the context clearly indicates otherwise.

The terminology and phraseology used herein is for the purpose of description and should not be regarded as limiting. The use of the terms "comprising", "containing", "having" and "including" and variations thereof used herein, are meant to encompass the items listed thereafter and equivalents thereof as well as additional items.

The present inventors postulated that the poxvirus particle is as good or better at presenting HIV-1 Env or a chimeric Env as a VLP or adjuvant. The earlier immunogenic response observed with the inclusion of Gag suggests that VLPs generated with Gag as a scaffold for Env elicit a more rapid neutralizing antibody response. The quality of the response was no different from Env presented on a VLP as opposed to presentation on a poxvirus.

The invention described herein relates to the use of a combination of modified, rationally designed, HIV-1 envelope proteins and the modified vaccinia Ankara (MVA) poxvirus to induced high titres of binding antibodies and neutralising antibodies.

For the present invention the HIV-1 envelope genes were modified in the following ways:

1 . The gene was codon optimised for expression in humans. The following items were also taken into account during the optimisation and modified where necessary: GC content, CpG dinucleotides content, mRNA secondary structure, cryptic splicing sites, premature PolyA sites, internal chi sites and ribosomal binding sites, negative CpG islands, RNA instability motif (ARE), repeat sequences (direct repeat, reverse repeat, and Dyad repeat), restriction sites that may interfere with cloning. Any potential poxvirus termination signals (TTTTTNT) were removed from the coding sequence and a Kozak sequence was included for optimal expression. In addition a poxvirus termination sequence was added directly after the stop codon (TGA) of the envelope gene.

2. The cytoplasmic domain was truncated to reduce gp160 to gp150.

3. The furin cleavage site was replaced with a flexible glycine linker region to allow for proper folding of the envelope protein in the absence of furin.

4. The I559P mutation was introduced to stabilize the gp41 trimer.

5. The native HIV-1 leader sequence was replaced with the tissue plasminogen activator leader sequence (tPA) to direct the envelope protein to the cell membrane, where, ultimately, it becomes incorporated into the poxvirus outer membrane.

6. A poxvirus early/late promoter (mH5) was added upstream of the modified env gene. This allows for expression both early, in the cytoplasm, where the envelope protein can be efficiently transported by means of the tPA leader to the cell membrane, as well as late, in the viral factories, where the envelope protein can be incorporated into the poxvirus virion.

In an alternative embodiment of the invention an alternative envelope design is also incorporated where chimeric HIV-lnfluenza envelope proteins were presented by MVA.

The HIV-1 envelope glycoprotein is present on the surface of the HIV-1 virion or virus-like particles at a very low density as compared to most other enveloped viruses. It has been shown that substitution of various subunits of HIV-1 Env with the corresponding elements from other viral glycoproteins can increase Env spike density on the cell membrane and surface of virus-like particles (VLPs) whilst retaining immunogenicity. Therefore, two different chimaeras were generated in which either the whole of HIV-1 gp41 was replaced with the corresponding influenza H5N1 HA₂ stalk (gp120HA2) or the membrane proximal external region (MPER) domain of gp41 was retained and only the transmembrane domain and cytoplasmic tail of HA₂ was used (gp140HA2tr). These chimeras contained the same modifications listed above for the HIV-1 envelope. To ensure stability of the recombinant poxvirus the env and chimeric env-HA₂ genes were inserted into a conserved region of MVA, between two transcriptionally convergent genes (I8R and G1 L). During budding of the mature virion the HIV-1 envelope protein is incorporated into the outer membrane of the poxvirion. The combination of the de novo produced HIV-1 envelope protein under the control of an early/late poxvirus promoter with the poxvirus results in well-presented envelope in the correct trimeric conformation on the surface of the poxvirus. The poxvirus has potent adjuvant properties and is highly immunogenic. The recombinant poxvirus presenting HIV-1 env on its surface is able to elicit a humoral (neutralizing) antibody response to protein entering the (animal) system. Once internalized the Env protein will be degraded via the proteosomal pathway and be presented on MHCII molecules to elicit an antibody response. Additionally, de novo expression of Env from the poxvirus will result in T cell help to further boost the humoral response.

This invention describes a potential vaccine candidate for HIV-1 . Additional envelope proteins from different HIV-1 strains could be modified in a similar manner; and different viral vectors (including different host-restricted poxviruses) could be used to express the HIV-1 envelope antigens.

This invention also describes a way of presenting chimeric Env proteins using a recombinant poxvirus as a vector, preferably the poxvirus is MVA.

As used herein, the term "MVA" or "modified vaccinia Ankara" refers to a highly attenuated strain of vaccinia virus having a sequence substantially identical to the sequence of GenBank Accession number U94848. MVA was initially developed as a poxvirus vaccine produced by more than 500 passages of vaccinia virus in chicken cells. Approximately 10% of the vaccinia genome is absent from MVA rendering it unable to replicate efficiently in primate cells and making MVA an ideal vector for clinical investigation for vaccination against other non-poxvirus diseases due to its high safety profile.

A "protein," "peptide" or "polypeptide" is any chain of two or more amino acids, including naturally occurring or non-naturally occurring amino acids or amino acid analogues, irrespective of post-translational modification (e.g., glycosylation or phosphorylation).

The terms "nucleic acid", "nucleic acid molecule" and "polynucleotide" are used herein interchangeably and encompass both ribonucelotides (RNA) and deoxyribonucleotides (DNA), including cDNA, genomic DNA, and synthetic DNA. The nucleic acid may be double-stranded or single-stranded. Where the nucleic acid is single-stranded, the nucleic acid may be the sense strand or the antisense strand. A nucleic acid molecule may be any chain of two or more covalently bonded nucleotides, including naturally occurring or non-naturally occurring nucleotides, or nucleotide analogs or derivatives. By "RNA" is meant a sequence of two or more covalently bonded, naturally occurring or modified ribonucleotides. The term "DNA" refers to a sequence of two or more covalently bonded, naturally occurring or modified deoxyribonucleotides.

The term "isolated", is used herein and means having been removed from its natural environment.

The term "purified", relates to the isolation of a molecule or compound in a form that is substantially free of contamination or contaminants. Contaminants are normally associated with the molecule or compound in a natural environment, purified thus means having an increase in purity as a result of being separated from the other components of an original composition. The term "purified nucleic acid" describes a nucleic acid sequence that has been separated from other compounds including, but not limited to polypeptides, lipids and carbohydrates which it is ordinarily associated with in its natural state.

The term "complementary" refers to two nucleic acids molecules, e.g., DNA or RNA, which are capable of forming Watson-Crick base pairs to produce a region of double-strandedness between the two nucleic acid molecules. It will be appreciated by those of skill in the art that each nucleotide in a nucleic acid molecule need not form a matched Watson-Crick base pair with a nucleotide in an opposing complementary strand to form a duplex. One nucleic acid molecule is thus "complementary" to a second nucleic acid molecule if it hybridizes, under conditions of high stringency, with the second nucleic acid molecule. A nucleic acid molecule according to the invention includes both complementary molecules.

As used herein a "substantially identical" sequence is an amino acid or nucleotide sequence that differs from a reference sequence only by one or more conservative substitutions, or by one or more non-conservative substitutions, deletions, or insertions located at positions of the sequence that do not destroy or substantially reduce the antigenicity of one or more of the expressed polypeptides or of the polypeptides encoded by the nucleic acid molecules. Alignment for purposes of determining percent sequence identity can be achieved in various ways that are within the knowledge of those with skill in the art. These include using, for instance, computer software such as ALIGN, Megalign (DNASTAR), CLUSTALW or BLAST software. Those skilled in the art can readily determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. In one embodiment of the invention there is provided for a polypeptide or polynucleotide sequence that has at least about 80% sequence identity, at least about 90% sequence identity, or even greater sequence identity, such as about 95%, about 96%, about 97%, about 98% or about 99% sequence identity to the sequences described herein.

Alternatively, or additionally, two nucleic acid sequences may be "substantially identical" if they hybridize under high stringency conditions. The "stringency" of a hybridisation reaction is readily determinable by one of ordinary skill in the art, and generally is an empirical calculation which depends upon probe length, washing temperature, and salt concentration. In general, longer probes required higher temperatures for proper annealing, while shorter probes require lower temperatures. Hybridisation generally depends on the ability of denatured DNA to re- anneal when complementary strands are present in an environment below their melting temperature. A typical example of such "stringent" hybridisation conditions would be hybridisation carried out for 18 hours at 65°C with gentle shaking, a first wash for 12 min at 65°C in Wash Buffer A (0.5% SDS; 2XSSC), and a second wash for 10 min at 65°C in Wash Buffer B (0.1 % SDS; 0.5% SSC).

Those skilled in the art will appreciate that polypeptides, peptides or peptide analogues can be synthesised using standard chemical techniques, for instance, by automated synthesis using solution or solid phase synthesis methodology. Automated peptide synthesisers are commercially available and use techniques known in the art. Polypeptides, peptides and peptide analogues can also be prepared from their corresponding nucleic acid molecules using recombinant DNA technology.

As used herein, the term "gene" refers to a nucleic acid that encodes a functional product, for instance an RNA, polypeptide or protein. A gene may include regulatory sequences upstream or downstream of the sequence encoding the functional product.

As used herein, the term "coding sequence" refers to a nucleic acid sequence that encodes a specific amino acid sequence. On the other hand a "regulatory sequence" refers to a nucleotide sequence located either upstream, downstream or within a coding sequence. Generally regulatory sequences influence the transcription, RNA processing or stability, or translation of an associated coding sequence. Regulatory sequences include but are not limited to: effector binding sites, enhancers, introns, polyadenylation recognition sequences, promoters, RNA processing sites, stem-loop structures, translation leader sequences;.

In some embodiments, the genes used in the method of the invention may be operably linked to other sequences. By "operably linked" is meant that the nucleic acid molecules encoding the recombinant env polypeptides of the invention and regulatory sequences are connected in such a way as to permit expression of the proteins when the appropriate molecules are bound to the regulatory sequences. Such operably linked sequences may be contained in vectors or expression constructs which can be transformed or transfected into host cells for expression. It will be appreciated that any vector or vectors can be used for the purposes of expressing the recombinant antigenic polypeptides of the invention.

The term "promoter" refers to a DNA sequence that is capable of controlling the expression of a nucleic acid coding sequence or functional RNA. A promoter may be based entirely on a native gene or it may be comprised of different elements from different promoters found in nature. Different promoters are capable of directing the expression of a gene in different cell types, or at different stages of development, or in response to different environmental or physiological conditions. A "constitutive promoter" is a promoter that direct the expression of a gene of interest in most host cell types most of the time.

The term "recombinant" means that something has been recombined. When used with reference to a nucleic acid construct the term refers to a molecule that comprises nucleic acid sequences that are joined together or produced by means of molecular biological techniques. The term "recombinant" when used in reference to a protein or a polypeptide refers to a protein or polypeptide molecule which is expressed from a recombinant nucleic acid construct created by means of molecular biological techniques. Recombinant nucleic acid constructs may include a nucleotide sequence which is ligated to, or is manipulated to become ligated to, a nucleic acid sequence to which it is not ligated in nature, or to which it is ligated at a different location in nature. Accordingly, a recombinant nucleic acid construct indicates that the nucleic acid molecule has been manipulated using genetic engineering, i.e. by human intervention. Recombinant nucleic acid constructs may be introduced into a host cell by transformation. Such recombinant nucleic acid constructs may include sequences derived from the same host cell species or from different host cell species. As used herein, the term "chimeric", means that a sequence comprises of sequences that have been "recombined". By way of example sequences are recombined and are not found together in nature. The term "recombine" or "recombination" refers to any method of joining two or more polynucleotides. The term includes end to end joining, and insertion of one sequence into another. The term is intended to include physical joining techniques, for instance, sticky-end ligation and blunt-end ligation. Sequences may also be artificially synthesized to contain a recombined sequence. The term may also encompass the integration of one sequence into a second sequence by way of, for example, homologous recombination.

The term "vector" refers to a means by which polynucleotides or gene sequences can be introduced into a cell. There are various types of vectors known in the art including plasmids, viruses, bacteriophages and cosmids. Generally polynucleotides or gene sequences are introduced into a vector by means of a cassette. The term "cassette" refers to a polynucleotide or gene sequence that is expressed from a vector, for example, the polynucleotide or gene sequences encoding recombinant MVA, DNA expression vector encoding a modified HIV-1 env and/or the soluble modified HIV-1 env protein of the invention of the invention. A cassette generally comprises a gene sequence inserted into a vector, which in some embodiments, provides regulatory sequences for expressing the polynucleotide or gene sequences. In other embodiments, the vector provides the regulatory sequences for the expression of the polypeptides of the invention. In further embodiments, the vector provides some regulatory sequences and the nucleotide or gene sequence provides other regulatory sequences. "Regulatory sequences" include but are not limited to promoters, transcription termination sequences, enhancers, splice acceptors, donor sequences, introns, ribosome binding sequences, poly(A) addition sequences, and/or origins of replication.

The recombinant modified vaccinia Ankara virus presenting HIV-1 envelope proteins or compositions of the invention containing the recombinant MVA can be provided either alone or in combination with other compounds (for example, nucleic acid molecules, small molecules, peptides, or peptide analogues), preferably the recombinant MVA is provided together with a DNA expression vector encoding a modified HIV-1 env and/or soluble modified HIV-1 env protein, in the presence of a liposome, an adjuvant, or any carrier, such as a pharmaceutically acceptable carrier and in a form suitable for administration to mammals, for example, humans, cattle, sheep, etc.

In one embodiment of the invention the recombinant MVA, DNA expression vector encoding a modified HIV-1 env and/or the soluble modified HIV-1 env protein of the invention is formulated for immunization together with an adjuvant. Adjuvants are well known to those of skill in the art of vaccine development and are not limited to the adjuvants specifically exemplified herein.

As used herein a "pharmaceutically acceptable carrier" or "excipient" includes any and all antibacterial and antifungal agents, coatings, dispersion media, solvents, isotonic and absorption delaying agents, and the like that are physiologically compatible. A "pharmaceutically acceptable carrier" may include a solid or liquid filler, diluent or encapsulating substance which may be safely used for the administration of the recombinant antigen or vaccine composition to a subject. The pharmaceutically acceptable carrier can be suitable for intramuscular, intradermal, intravenous, intraperitoneal, subcutaneous, oral or sublingual administration. Pharmaceutically acceptable carriers include sterile aqueous solutions, dispersions and sterile powders for the preparation of sterile solutions. The use of media and agents for the preparation of pharmaceutically active substances is well known in the art. Where any conventional media or agent is incompatible with the active compound, use thereof in the pharmaceutical compositions of the invention is not contemplated. Supplementary active compounds can also be incorporated into the compositions.

Suitable formulations or compositions to administer the recombinant MVA and compositions (including a DNA expression vector encoding a modified HIV-1 env and/or soluble modified HIV-1 env protein) to subjects infected with HIV or subjects which are presymptomatic for a condition associated with HIV infection fall within the scope of the invention. Any appropriate route of administration may be employed, such as, parenteral, intravenous, intradermal, subcutaneous, intramuscular, intracranial, intraorbital, ophthalmic, intraventricular, intracapsular, intraspinal, intrathecal, intracistemal, intraperitoneal, intranasal, aerosol, topical, or oral administration.

For vaccine formulations and pharmaceutical compositions, an effective amount of the recombinant MVA or compositions (including a DNA expression vector encoding a modified HIV-1 env and/or soluble modified HIV-1 env protein) of the invention can be provided, either alone or in combination with other compounds, with immunological adjuvants, for example, aluminium hydroxide dimethyldioctadecyl- ammonium hydroxide or Freund's incomplete adjuvant. The recombinant MVA or compositions (including a DNA expression vector encoding a modified HIV-1 env and/or soluble modified HIV-1 env protein) of the invention may also be linked with suitable carriers and/or other molecules, such as bovine serum albumin or keyhole limpet haemocyanin in order to enhance immunogenicity.

Vaccine formulations and compositions that are useful in the present invention include the recombinant MVA, the DNA expression vector encoding a modified HIV-1 env and/or soluble modified HIV-1 env protein that prime and/or boost an immune response to HIV.

In one embodiment, the HIV-1 antigens are capable of "priming" an immune response to HIV. Examples of such priming compositions include the recombinant MVA of the invention, DNA expression vector encoding a modified HIV-1 env of the invention and/or soluble modified HIV-1 env protein of the invention, these compositions prime an immune response to HIV.

It will further be appreciated that a "boost" composition may include HIV-1 antigens which are administered to the subject in two or more doses after the initial priming inoculation. The boosting composition may include at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine or at least ten subsequent inoculations with at least two HIV-1 antigens selected from the group consisting of the recombinant MVA, the DNA expression vector encoding a modified HIV-1 env and/or soluble modified HIV-1 env protein.

In some embodiments, the recombinant MVA or compositions (including a DNA expression vector encoding a modified HIV-1 env and/or soluble modified HIV-1 env protein) according to the invention may be provided in a kit, optionally with a carrier and/or an adjuvant, together with instructions for use.

An "effective amount" of a recombinant MVA or composition (including a DNA expression vector encoding a modified HIV-1 env and/or soluble modified HIV-1 env protein) according to the invention includes a therapeutically effective amount, immunologically effective amount, or a prophylactically effective amount. A "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result, such as treatment of an infection or a condition associated with such infection. The outcome of the treatment may for example be measured by a decrease in viraemia, inhibition of viral gene expression, delay in development of a pathology associated with HIV infection, stimulation of the immune system, or any other method of determining a therapeutic benefit. A therapeutically effective amount of a compound may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the compound to elicit a desired response in the individual. Dosage regimens may be adjusted to provide the optimum therapeutic response. A therapeutically effective amount is also one in which any toxic or detrimental effects of the compound are outweighed by the therapeutically beneficial effects.

The dosage of the recombinant MVA or compositions (including a DNA expression vector encoding a modified HIV-1 env and/or soluble modified HIV-1 env protein) of the present invention will vary depending on the symptoms, age and body weight of the subject, the nature and severity of the disorder to be treated or prevented, the route of administration, and the form of the composition. Any of the compositions of the invention may be administered in a single dose or in multiple doses. The dosages of the compositions of the invention may be readily determined by techniques known to those of skill in the art or as taught herein.

By "immunogenically effective amount" is meant an amount effective, at dosages and for periods of time necessary, to achieve a desired immune response. The desired immune response may include stimulation or elicitation of an immune response, for instance a T-cell response.

A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired prophylactic result, such as prevention of onset of a condition associated with HIV infection. Typically, a prophylactic dose is used in a subject prior to or at an earlier stage of disease, so that a prophylactically effective amount may be less than a therapeutically effective amount.

Dosage values may vary and be adjusted over time according to the individual need and the judgment of the person administering or supervising the administration of the recombinant MVA or compositions (including a DNA expression vector encoding a modified HIV-1 env and/or soluble modified HIV-1 env protein) of the invention. Dosage ranges set forth herein are exemplary only and do not limit the dosage ranges that may be selected. The amount of active compound(s) in the composition may vary according to factors such as the disease state, age, sex, and weight of the individual. Dosage regimens may be adjusted to provide the optimum therapeutic response. For example, a single dose may be administered, or multiple doses may be administered over time. It may be advantageous to formulate the compositions in dosage unit forms for ease of administration and uniformity of dosage.

The vaccination protocol for eliciting an immune response against HIV-1 in a subject as defined herein typically comprises a series of single doses of the HIV-1 immunogens described herein. A single dose or dosage, as used herein, refers to the priming dose (i.e. initial first or second dose with the same immunogen), and any subsequent dose, respectively, which are preferably administered in order to "boost" the immune reaction. In this context, each single dosage comprises the administration of one of the HIV-1 immunogens or antigens according to the invention, wherein the interval between the administration of two single dosages can vary from at least one week, preferably 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 or 12 weeks apart. Most preferably, the HIV-1 immunogens or compositions of the invention are administered at intervals of either 4 or 8 weeks apart. It will be appreciated that the intervals between single dosages may be constant or vary over the course of the immunization protocol, e.g. the intervals may be shorter in the beginning (such as 4 weeks apart) and longer towards the end of the protocol (such as 8 weeks apart). Additionally, depending on the total number of single dosages and the interval between single dosages, the immunization protocol may extend over a period of time, which preferably lasts at least one week, more preferably several weeks, even more preferably several months (e.g. 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 18 or 24 months). Each single dosage encompasses the administration of one of the HIV-1 immunogens described herein.

The term "preventing", when used in relation to an infectious disease, or other medical disease or condition, is well understood in the art, and includes administration of a composition which reduces the frequency of or delays the onset of symptoms of a condition in a subject relative to a subject which does not receive the composition. Prevention of a disease includes, for example, reducing the number of diagnoses of the infection in a treated population versus an untreated control population, and/or delaying the onset of symptoms of the infection in a treated population versus an untreated control population.

The term "prophylactic or therapeutic" treatment is well known to those of skill in the art and includes administration to a subject of one or more of the compositions of the invention. If the composition is administered prior to clinical manifestation of the unwanted condition (e.g., disease or other unwanted state of the subject) then the treatment is prophylactic, i.e., it protects the host against developing the unwanted condition, whereas if it is administered after manifestation of the unwanted condition, the treatment is therapeutic (i.e., it is intended to diminish, ameliorate, or stabilise the existing unwanted condition or side effects thereof).

Toxicity and therapeutic efficacy of compositions of the invention may be determined by standard pharmaceutical procedures in cell culture or using experimental animals, such as by determining the LD₅₀ and the ED₅₀. Data obtained from the cell cultures and/or animal studies may be used to formulate a dosage range for use in a subject. The dosage of any composition of the invention lies preferably within a range of circulating concentrations that include the ED₅₀ but which has little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilised. For compositions of the present invention, the therapeutically effective dose may be estimated initially from cell culture assays.

The following examples are offered by way of illustration and not by way of limitation.

EXAMPLE 1

Antigen Design

Modified HIV Env

Two different antigens were used. The first HIV-1 envelope sequence used (SEQ ID NO:1 ) was taken from the superinfecting virus from participant CAP256 in the CAPRISA 002 Acute infection cohort (Doria-Rose et al., 2014; Moore et al., 2013). The coding sequence of this gp160 envelope (SEQ ID NO:2) from (clone 256.2.06. c7) was provided by Dr Penny Moore (Senior Medical Scientist, Centre for HIV and STIs, National Institute for Communicable Diseases, Johannesburg). The Du151 envelope amino acid sequence (SEQ ID NO:13) and nucleotide sequence (SEQ ID NO:14) were retrieved from GenBank (Accession number AF544008.1 ). Envelope antigens were designed based on the native flexible linker approach to enable the production of native-like trimers in the absence of furin cleavage. The native HIV Env cleavage site was replaced with a 10 amino acid flexible linker comprising of 2 repeats of the Glycine-Serine based (GGGGS) motif (SEQ ID NO:23). The isoleucine at residue 559 in the N-terminal heptad repeat of gp41 was mutated to a proline. For MVA expression the coding sequence prematurely terminated by the introduction of a stop codon to generate gp150 and for production of soluble protein a stop codon was introduced to generate gp140. The modified CAP256 gp150 has the amino acid sequence of SEQ ID NO:3 and the modified Du151 gp150 has the amino acid sequence of SEQ ID NO:15. The modified CAP256 gp140 has the amino acid sequence of SEQ ID NO:5 and the modified Du151 gp140 has the amino acid sequence of SEQ ID NO:17. The HIV-1 envelope gene was codon optimised for expression in humans. The following items were also taken into account during the optimisation and modified where necessary: GC content, CpG dinucleotides content, mRNA secondary structure, cryptic splicing sites, premature PolyA sites, internal chi sites and ribosomal binding sites, negative CpG islands, RNA instability motif (ARE), repeat sequences (direct repeat, reverse repeat, and Dyad repeat), restriction sites that may interfere with cloning. Any potential poxvirus termination signals (TTTTTNT) were removed from the coding sequence and a Kozak sequence was included for optimal expression. In addition a poxvirus termination sequence was added directly after the stop codon (TGA) of the envelope gene. The native HIV-1 envelope leader sequence was replaced with the tissue plasminogen activator leader sequence. The modified CAP256 gp150 has the nucleotide sequence of SEQ ID NO:4 and the modified Du151 gp150 has the nucleotide sequence of SEQ ID NO:16. The modified CAP256 gp140 has the nucleotide sequence of SEQ ID NO:6 and the modified Du151 gp140 has the nucleotide sequence of SEQ ID NO:18.

Chimeric HIV-HA Env

Two different chimaeras were generated in which either the whole of HIV-1 gp41 was replaced with the corresponding influenza HA₂ stalk (gp120HA2) or the MPER domain of gp41 was retained and only the transmembrane domain and cytoplasmic tail of HA₂ was used (gp140HA2tr) (Figure 1 ). As for the HIV-1 gp150, the native HIV Env cleavage site was replaced with a 10 amino acid flexible linker comprising of 2 repeats of the Glycine-Serine based (GGGGS) motif (SEQ ID NO:23). The isoleucine at residue 559 in the N-terminal heptad repeat of HIV-1 gp140HA2tr was mutated to a proline. The modified CAP256 gp120HA2 has the amino acid sequence of SEQ ID NO:7 and the modified Du151 gp120HA2 has the amino acid sequence of SEQ ID NO:19. The modified CAP256 gp140HA2tr has the amino acid sequence of SEQ ID NO:9 and the modified Du151 gp140HA2tr has the amino acid sequence of SEQ ID NO:21 . The HIV-1 envelope gene was codon optimised for expression in humans. The following items were also taken into account during the optimisation and modified where necessary: GC content, CpG dinucleotides content, mRNA secondary structure, cryptic splicing sites, premature PolyA sites, internal chi sites and ribosomal binding sites, negative CpG islands, RNA instability motif (ARE), repeat sequences (direct repeat, reverse repeat, and Dyad repeat), restriction sites that may interfere with cloning. Any potential poxvirus termination signals (TTTTTNT) were removed from the coding sequence and a Kozak sequence was included for optimal expression. In addition a poxvirus termination sequence was added directly after the stop codon (TGA) of the envelope gene. The native HIV-1 envelope leader sequence was replaced with the tissue plasminogen activator leader sequence. The modified CAP256 gp120HA2 has the nucleotide sequence of SEQ ID NO:8 and the modified Du151 gp120HA2 has the nucleotide sequence of SEQ ID NO:20. The modified CAP256 gp140HA2tr has the nucleotide sequence of SEQ ID NO:10 and the modified Du151 gp140HA2tr has the nucleotide sequence of SEQ ID NO:22.

EXAMPLE 2

Construction of recombinant MVA expressing different Env and chimeric Env constructs

Transfer vector construction

Generally poxvirus transfer vectors comprise foreign genes under the control of a poxvirus promoter flanked by poxvirus sequences for insertion into the poxvirus genome. A reporter gene under the control of a poxvirus promoter can be included as well as genes coding for selection of the recombinant. These are standard components of a poxvirus transfer vector.

The transfer vector depicted in Figure 2 was constructed by cloning the HIV-1 envelope genes into the H/ndlM and EcoRI restriction enzyme sites of plasmid pSSPEx (SEQ ID NO:24).

Isolation of recombinant MVA

BHK-21 cells were infected with wild type MVA as retrieved from GenBank (Accession number U94848) or MVA-Gag containing a HIV-1 subtype C mosaic Gag protein (SEQ ID NO:1 1 ) encoded by the gag gene (SEQ ID NO:12)at an MOI of 0.01 or 0.1 and transfected with 2 or 3 μg of transfer vector in a total volume of 1 ml in a 12-well plate. 3 days post infection the cells were lysed by three cycles of freezing and thawing. This lysate was passaged on RK13 cells for selection of virus expressing the K1 L gene. Fluorescing viral foci were purified by serial dilution and virus from single foci were bulked up in RK13 cells, in a series of larger wells and flasks. From a seed stock a working stock was prepared in hyperflasks. Virus from hyperflasks was purified by lysis of the cells by freezing and thawing three times, followed by low speed centrifugation to remove cell debris and then high speed centrifugation (47 000 rcf) through a cushion of 36 % sucrose in PBS. The viral pellet was resuspended in a small volume of PBS +10 % glycerol and titrated in RK13 cells by counting fluorescing foci in wells infected with a serial 10-fold dilution of the virus.

EXAMPLE 3

Construction of recombinant DNA expressing different Env and chimeric Env constructs

HIV-1 envelope genes and chimeras were cloned into H/ndlM and EcoR\ restriction enzyme sites of pTHCapR, an expression vector containing a porcine circovirus enhancer element, which was chosen for superior expression and immunogenicity over commercial plasmids (Tanzer et al. (201 1 )). This mammalian expression plasmid backbone was renamed pMExT for Mammalian Expression with tPA leader.

EXAMPLE 4

Expression of soluble HIV-1 envelope protein

Stable cell lines were generated for both CAP256SU and Du151 gp140 expression. An inter-ribosomal entry site (Ires) and neomycin resistance gene (NeoR) was introduced directly behind the stop codon of gp140 in an expression vector (Tanzer et al. (201 1 )). This will result in RNA transcription of Env and NeoR from the same promoter, linking protein expression of gp140 from the promoter to NeoR from Ires. T75 flasks with HEK293 cells were grown to confluency and transfected with these constructs. The next day, cells were passaged into T150 flasks and geneticin (600 μg/ml) was added to the medium for selection of NeoR. After passaging cells for≥10 rounds (P10), cells are considered to show stable expression of the transgenes. The cells were then grown in HyperFlasks and by coating these flasks with poly-l-lysine, 3-4 repeat harvests were feasible from a single HyperFlask. For repeat harvesting, flasks were cycled between serum-free medium and medium + foetal calf serum (FCS). The media containing secreted Env was harvested and cleared of cellular debris by a short, low speed spin. Cleared media was slowly pumped over a column containing agarose-conjugated lectin Galanthus nivalis which binds proteins with high mannose content such as Env. After media was passed through, the column was washed with PBS + 0.5 M NaCI followed by PBS. The Env protein was eluted from the column using PBS + 1 M methyl aD-manno- pyranoside and concentrated by centrifugation using Vivaspin columns. Concentrated, eluted Env protein eluted from the lectin column, was injected into a Superdex 200 HiLoad 16/600 column for Size Exclusion Chromatography (SEC). The fractions containing trimeric protein were collected and characterized on a non- denaturing protein gel before pooling.

EXAMPLE 5

Immunoqenicitv results

Protein Immunisation Experiment

Rabbit immunizations and blood sampling was conducted at the University of Cape Town, in accordance with the guidelines and approval of the appropriate ethics committee (AEC 014-030 & 015-051 ). Three month old New Zealand white rabbits were immunized with 40 μg of recombinant protein suspended in 2% Alhydrogel® Adjuvant (Invivogen) at a concentration of 1 :1 (antigen: adjuvant). Groups of 5 rabbits were immunized intramuscularly into the quadriceps muscle of the hind leg. Blood was drawn 4 weeks after immunization.

MVA prime, protein boost immunogenicity experiment using HIV-1 CAP256 SU envelope

Rabbits were inoculated with MVA that expressed HIV-1 Env or Env-HA₂ chimaeras and the mosaic HIV-1 subtype C Gag. MVA expressing HIV-1 CAP256 gp150 alone (without Gag) was used as a control to analyse the effect of Gag on the immune response (MVAC5). After immunisation with 10⁸ pfu MVA at weeks 0 and 4, rabbits were boosted at weeks 12, 20 & 28 with 40 μg of trimeric, soluble gp140 protein suspended in 2% Alhydrogel® Adjuvant (Invivogen) at a concentration of 1 :1 (antigen: adjuvant).

MVA prime, protein boost using HIV- 1 Du151 envelope

In this experiment the MVA prime protein boost strategy, used in the previous experiment, was repeated with MVA expressing HIV-1 Du151 Env rather than CAP256SU and HIV-1 Du151 gp140 soluble protein. An additional group of rabbits was included that was primed with SAAVI MVA-C (expresses a polyprotein containing Gag, RT, Tat & Net and GP150 (Du151 )) and boosted with soluble HIV-1 Du151 gp140 protein.

DNA x 2, MVA x 2, protein x 2 using HIV-1 CAP256 SU envelope

Rabbits were inoculated at weeks 0 and 4 with 100 μg DNA that expressed HIV-1 Env or Env-HA₂ chimaeras and 100 μg DNA that expressed mosaic HIV-1 subtype C Gag. DNA expressing HIV-1 CAP256 gp150 alone (without Gag) was used as a control to analyse the effect of Gag on the immune response (DNAC5). Rabbits were then boosted with 10⁸ pfu of the matching MVA vaccines at weeks 8 and 12 and then further boosted at weeks 20 & 28 with 40 μg of trimeric, soluble gp140 protein suspended in 2% Alhydrogel® Adjuvant (Invivogen) at a concentration of 1 :1 (antigen: adjuvant).

Binding EL ISA

To assess Env binding antibody titres in rabbit sera, ELISA experiments were performed. Nunc MaxiSorp® flat-bottom 96 well plates (Sigma) were coated overnight with 10 ng/well HIV-1 envelope protein at 4 °C. ELISA plates were washed with PBST (PBS containing 0.1 % Tween 20) and blocked using 5% non-fat milk PBST. Rabbit sera was used in the primary incubation in a serial dilution range starting at 1 :10 in 5 % non-fat milk PBST. Detection antibody used was anti-rabbit IgG HRP (1 :10000) (Roche). ELISAs for the whole time course and all groups were performed at the same time on duplicate plates. Antibody end-point titres were calculated from 4PL curves of duplicate data points with the threshold set as twice the geometric mean of the ELISA signal over the whole, matching pre-bleed serial dilution range. Data plotted as mean +/- SEM for whole group.

Pseudovihon cell entry neutralization assays

Neutralization was measured as a reduction in luciferase gene expression after a single round of infection of JC53bl-13 cells, also known as TZM-bl cells (NIH AIDS Research and Reference Reagent Program), with Env-pseudotyped viruses (MW965.26, MN.3, 6644, CA146, 1 107356, CAP37, CT349, Du156, 188146, CAP256.SU, ZM53, Ce1086). Titer was calculated as the reciprocal plasma/serum dilution causing a 50 % reduction of relative light units (ID₅₀). These assays were carried out by Lynne Morris's group at the NICD, Johannesburg. Results

No obvious differences were seen in pseudovirion cell entry neutralization between the different MVA primed animals expressing the CAP256SU envelope or its chimeras (Table 1 ). MVA priming resulted in some Tier 1 B neutralization breadth (6644 & 1 107356). Most importantly, MVA primed rabbits developed autologous Tier2 neutralization in 50 % of animals, with IC50 neutralization values ranging from 1 :23 to 333.

Including mosaic Gag in the MVA prime does not appear to affect binding antibody titres or final neutralization titres. However, Tier 2 neutralization was observed with MVA priming after the first protein boost when mosaic Gag was present but not in the absence of Gag. Western blot analysis of purified virus and Virus-like particles (VLPs) showed that both envelope and Gag proteins are present in the virion particles and VLPs respectively.

Interestingly, there is a significant correlation between the development of Tier 2 neutralization and lower serum Env binding titres for animals primed with MVA expressing CAP256 gp150 and mosaic Gag (MVAGC5) and MVA only expressing gp150 (MVAC5) (Figure 3).

Data showed high titres of binding antibodies to Env were elicited after a single inoculation of MVA expressing Du151 Env and mosaic Gag (MVAGD5), whereas no detectable binding antibodies to Env were elicited after 2 inoculations of SAAVI MVA-C (which expresses a polyprotein of Gag, RT, Tat & Nef and HIV-1 Du151 gp150) and could only be detected after 2 doses of SAAVI MVA-C and the 1 st protein boost at week 12 (Figure 4). This demonstrates a clear improvement on the previous SAAVI vaccine tested in clinical trials. The envelope protein is based on the same sequence in each vaccine but the Env in MVAGD5 has been further modified (native leader replaced with tPA leader sequence, furin cleavage site replaced with 10 amino acid linker, I559P mutation included). In addition MVAGD5 expresses the HIV-1 subtype C mosaic Gag which assembles into virus like particles, whereas SAAVI-MVA C expresses the Gag from HIV-1 strain Du422 and the myristylation signal has been removed so it cannot produce VLPs. Table 1 : Neutralization titres of sera from rabbits that have received two rMVA inoculations and three protein boosts. MVA at weeks 0 and 4, boosted at weeks 12, 20 & 28 with trimeric, soluble CAP256 gp140

EXAMPLE 6

Comparison of responses with DNA prime vs MVA prime vs protein alone MVA expressing the modified Env induces binding antibody responses before the protein boost whereas this is not seen in the DNA prime experiments (Figure 5). There is no difference in the binding antibody titres between rabbits vaccinated with MVA or protein alone (Figure 5, week 8 & 12), indicating that the MVA prime alone is able to elicit high binding antibody titres. This implies that our novel MVA expressing the various versions of Env induces antibodies as well as the protein vaccines. This is a major advantage as MVA is much cheaper to produce than subunit protein vaccines. In addition, rabbits vaccinated with a MVA prime-protein boost regimen produced Tier 2 neutralizing antibodies whereas rabbits vaccinated with protein alone did not (Figure 6, Table 1 ).

EXAMPLE 7

DNAGC5 and MVAGC5 vaccination alone elicits Tier 2 autologous neutralizing antibodies

Rabbits inoculated with DNAGC5 at weeks 0 and 4, followed by MVAGC5 at weeks 8 and 12 and CAP256 gp140 trimeric protein at weeks 20 and 28 developed even better Tier 2 neutralizing response (4/5 rabbits) than rabbits vaccinated with 2 x MVA and 2 x protein, with neutralizing antibodies appearing after the second MVA boost (3/5 rabbits, Table 2). If Gag was included in the DNA and MVA vaccines autologous Tier 2 neutralizing antibodies were detected after the 2nd MVA inoculation whereas if rabbits were only vaccinated with DNA and MVA expressing gp150 Tier 2 neutralizing antibodies were only detected after the 1 st protein boost.

Table 2: Neutralization titres of sera from rabbits that have received two DNA, two MVA inoculations and two protein boosts. DNA at weeks 0 and 4, MVA at weeks 8 and 12 and soluble, trimeric CAP256 gp140 protein weeks 20 & 28.

REFERENCES

Capucci S, Wee EG, Schiffner T, LaBranche CC, Borthwick N, Cupo A, Dodd J, Dean H, Sattentau Q, Montefiori D, Klasse PJ, Sanders RW, Moore JP, Hanke T. 2017. HIV-1 -neutralizing antibody induced by simian adenovirus- and poxvirus MVA- vectored BG505 native-like envelope trimers. PLoS One 12:e0181886.

Tanzer, F.L., S ephard, E., Palmer, K. E., Burger, M., Williamson, A-L, Rybicki, E. P. 201 1 . The porcine circovirus type 1 capsid gene promoter improves antigen expression and immunogenicity in a HIV-1 plasmid vaccine. Virol J. 8: 51 .

Townsley S, Mohamed Z, Guo W, McKenna J, Cleveland B, LaBranche C, Beaumont D, Shen X, Yates NL, Pinter A, Tomaras GD, Ferrari G, Montefiori DC, Hu SL. 2016. Induction of Heterologous Tier 2 HIV-1 -Neutralizing and Cross- Reactive V1/V2-Specific Antibodies in Rabbits by Prime-Boost Immunization. J Virol 90:8644- 8660.

Claims

1 . A prime-boost vaccine comprising a priming HIV-1 immunogen and one or more boosting HIV-1 immunogens, wherein the priming HIV-1 immunogen comprises either: a) a DNA expression vector capable of expressing a modified HIV-1 gp150 envelope protein in a mammalian cell; or

b) a recombinant modified vaccinia Ankara (MVA) virus, including a nucleic acid encoding a modified HIV-1 gp150 envelope protein; and wherein the one or more boosting HIV-1 immunogens comprises : a) a recombinant modified vaccinia Ankara (MVA) virus, including a nucleic acid encoding a modified HIV-1 gp150 envelope protein; and/or b) a soluble modified HIV-1 gp140 envelope protein.

2. The vaccine of claim 1 , wherein the modified HIV-1 gp150 envelope protein comprises a truncated cytoplasmic domain, a flexible glycine linker replacing the furin cleavage site and an I559P mutation.

3. The vaccine of claim 1 or 2, wherein the modified HIV-1 gp140 envelope protein comprises a flexible glycine linker replacing the furin cleavage site and an I559P mutation.

4. The vaccine of any one of claims 1 to 3, wherein the MVA or DNA expression vector optionally includes a nucleic acid encoding an HIV-1 Gag protein.

5. The vaccine of any one of claims 1 to 4, wherein the modified HIV-1 gp150 envelope protein is substantially identical to the sequence of SEQ ID NO:3 or SEQ ID NO:15.

6. The vaccine of any one of claims 1 to 5, wherein the modified HIV-1 gp140 envelope protein is substantially identical to the sequence of SEQ ID NO:5 or SEQ ID NO:17.

7. The vaccine of any one of claims 1 to 5, wherein the nucleic acid encoding the modified HIV-1 gp150 envelope protein includes a tissue plasminogen activator leader sequence.

8. The vaccine of any one of claims 1 to 7, wherein the priming HIV-1 immunogen is the DNA expression vector capable of expressing a modified HIV-1 gp150 envelope protein in the mammalian cell; and

wherein a first boosting HIV-1 immunogen comprises the recombinant modified vaccinia Ankara (MVA) virus, including a nucleic acid encoding a modified HIV-1 gp150 envelope protein,

wherein a second boosting HIV-1 immunogen comprises the soluble modified HIV-1 gp140 envelope protein; and

wherein one or more consecutive doses of each priming HIV-1 immunogen and each boosting HIV-1 immunogen are administered.

9. The vaccine of any one of claims 1 to 8, wherein the modified HIV-1 envelope protein is capable of folding into a trimeric conformation.

10. A prime-boost vaccine for use in a method of inducing an immune response to HIV-1 in a subject, the method comprising administering one or more consecutive doses of a priming HIV-1 immunogen and one or more consecutive doses of a boosting HIV-1 immunogen to the subject, wherein the priming HIV-1 immunogen comprises either: a) a DNA expression vector capable of expressing a modified HIV-1 gp150 envelope protein in the subject; or

b) a recombinant modified vaccinia Ankara (MVA) virus, including a nucleic acid encoding a modified HIV-1 gp150 envelope protein; and wherein the boosting HIV-1 immunogen comprises: a) a recombinant modified vaccinia Ankara (MVA) virus, including a nucleic acid encoding a modified HIV-1 gp150 envelope protein; and/or b) a soluble modified HIV-1 gp140 envelope protein.

1 1 . The vaccine for use of claim 10, wherein the modified HIV-1 gp150 envelope protein comprises a truncated cytoplasmic domain, a flexible glycine linker replacing the furin cleavage site and an I559P mutation.

12. The vaccine for use of claim 10 or 1 1 , wherein the modified HIV-1 gp140 envelope protein comprises a flexible glycine linker replacing the furin cleavage site and an I559P mutation.

13. The vaccine for use of any one of claims 10 to 12, wherein the MVA or DNA expression vector optionally includes a nucleic acid encoding an HIV-1 Gag protein.

14. The vaccine for use of any one of claims 10 to 13, wherein the modified HIV-1 gp150 envelope protein is substantially identical to the sequence of SEQ ID NO:3 or SEQ ID NO:15.

15. The vaccine for use of any one of claims 10 to 13, wherein the modified HIV-1 gp140 envelope protein is substantially identical to the sequence of SEQ ID NO:5 or SEQ ID NO:17.

16. The vaccine for use of any one of claims 10 to 15, wherein the nucleic acid encoding the modified HIV-1 gp150 envelope protein includes a tissue plasminogen activator leader sequence.

17. The vaccine for use of any one of claims 10 to 16, wherein the priming HIV-1 immunogen is the DNA expression vector capable of expressing a modified HIV-1 gp150 envelope protein in the subject; and

wherein a second boosting HIV-1 immunogen comprises the soluble modified HIV-1 gp140 envelope protein, and wherein one or more consecutive doses of each priming HIV-1 immunogen and each boosting HIV-1 immunogen are administered to the subject.

18. The vaccine for use of any one of claims 10 to 17, wherein the immune response is a neutralising antibody response or a cytotoxic T lymphocyte response.

19. The vaccine for use of any one of claims 10 to 18, wherein the modified HIV-1 envelope protein is capable of folding into a trimeric conformation.

20. The vaccine for use of any one of claims 10 to 19, wherein the subject is a human.

21 . A method of inducing an immune response to HIV-1 in a subject, the method comprising administering one or more consecutive doses of a priming HIV-1 immunogen and one or more consecutive doses of a boosting HIV-1 immunogen to the subject, wherein the priming HIV-1 immunogen comprises either: a) a DNA expression vector capable of expressing a modified HIV-1 gp150 envelope protein in the subject; or

b) a recombinant modified vaccinia Ankara (MVA) virus, including a nucleic acid encoding a modified HIV-1 gp150 envelope protein; and wherein the boosting HIV-1 immunogen comprises: a) a recombinant modified vaccinia Ankara (MVA) virus, including a nucleic acid encoding a modified HIV-1 gp150 envelope protein; and/or

b) a soluble modified HIV-1 gp140 envelope protein.

22. The method of claim 21 , wherein the modified HIV-1 gp150 envelope protein comprises a truncated cytoplasmic domain, a flexible glycine linker replacing the furin cleavage site and an I559P mutation.

23. The method of claim 21 or 22, wherein the modified HIV-1 gp140 envelope protein comprises a flexible glycine linker replacing the furin cleavage site and an I559P mutation.

24. The method of any one of claims 21 to 23, wherein the MVA or DNA expression vector optionally includes a nucleic acid encoding an HIV-1 Gag protein.

25. The method of any one of claims 21 to 24, wherein the modified HIV-1 gp150 envelope protein is substantially identical to the sequence of SEQ ID NO:3 or SEQ ID NO:15.

26. The method of any one of claims 21 to 24, wherein the modified HIV-1 gp140 envelope protein is substantially identical to the sequence of SEQ ID NO:5 or SEQ ID NO:17.

27. The method of any one of claims 21 to 26, wherein the nucleic acid encoding the modified HIV-1 gp150 envelope protein includes a tissue plasminogen activator leader sequence.

28. The method of any one of claims 21 to 27, wherein the priming HIV-1 immunogen is the DNA expression vector capable of expressing a modified HIV-1 gp150 envelope protein in the subject; and

wherein a second boosting HIV-1 immunogen comprises the soluble modified HIV-1 gp140 envelope protein, and

wherein one or more consecutive doses of each priming HIV-1 immunogen and each boosting HIV-1 immunogen are administered to the subject.

29. The method of any one of claims 21 to 28, wherein the immune response is a neutralising antibody response or a cytotoxic T lymphocyte response.

30. The method of any one of claims 21 to 29, wherein the modified HIV-1 envelope protein is capable of folding into a trimeric conformation.

31 . The method of any one of claims 21 to 30, wherein the subject is a human.

32. A kit for inducing an immune response to HIV-1 infection in a subject comprising a priming composition, and at least one boosting composition; wherein the priming composition comprises an HIV-1 immunogen selected from either: a) a DNA expression vector capable of expressing a modified HIV-1 gp150 envelope protein in the subject; or

b) a recombinant modified vaccinia Ankara (MVA) virus, including a nucleic acid encoding a modified HIV-1 gp150 envelope protein; and wherein the at least one boosting composition comprises an HIV-1 immunogen selected from: a) a recombinant modified vaccinia Ankara (MVA) virus, including a nucleic acid encoding a modified HIV-1 gp150 envelope protein; and/or

b) a soluble modified HIV-1 gp140 envelope protein, and instructions for use to administer one or more consecutive doses of each priming composition and each boosting HIV-1 composition the subject.