WO2013140169A1

WO2013140169A1 - Immunogenic composition and methods of use thereof

Info

Publication number: WO2013140169A1
Application number: PCT/GB2013/050726
Authority: WO
Inventors: Anna-Lena Spetz-Holmgren; Guillaume Stewart-Jones
Original assignee: Isis Innovation Limited
Priority date: 2012-03-20
Filing date: 2013-03-20
Publication date: 2013-09-26

Abstract

The invention provides a composition for use in inducing an immune response to a first virus, wherein the composition comprises an immunogen derivable from a second virus and wherein the immunogen derivable from the second virus has between 30% and 90% protein sequence identity to an equivalent immunogen derivable from the first virus. The invention further comprises a method of inducing an immune response to a first virus, comprising administering to a subject an immunogenic composition comprising an immunogen derivable from a second virus and wherein the immunogen derivable from the second virus has between 30% and 90% protein sequence identity to an equivalent immunogen derivable from the first virus.

Description

IMMUNOGENIC COMPOSITION AND METHODS OF USE THEREOF

The invention relates to immunogenic compositions and the use of such compositions to generate an improved immune response in a subject.

HIV- 1 has infected approximately 60 million people and claimed approximately 25 million lives and history has shown a vaccine would be the most effective way of curtailing the pandemic. The socio-economic impact of this disease has so far been enormous, and it continues to spread, despite education, treatments and other interventions worldwide . Approximately 2 million people worldwide are infected each year and for each individual who is put onto antiretroviral therapy, 4 individuals become infected, underscoring the urgent and important need to develop a safe and effective HIV- 1 vaccine. Although HIV-2 is not as widespread or virulent as HIV-2 it would also be helpful to develop a vaccine against HIV-2.

Components of the Env protein of HIV are usually the preferred target for vaccines against HIV because antibodies raised against the envelope proteins can target HIV virus particles. In order for a vaccine to be effective against HIV it must target a conserved site, for example a conserved part of an Envelope protein, because HIV has a high mutation rate and the target sites change rapidly. The high mutation rate also leads to many strains of HIV being present within a population and even within an infected individual. During the last few years, considerable knowledge has been gained about the binding sites for antibodies capable of neutralizing a broad range of HIV- 1 strains. However, there is still a gap in the knowledge of how to focus the B-cell responses to vulnerable conserved sites within the HIV- 1 Envelope proteins. One immunological problem is that the variable parts of the virus are more immunogenic than the conserved parts.

It is an aim of the present invention to provide an immunogenic composition that is able to raise neutralising antibodies against a virus, for example HIV- 1. In a first aspect the present invention provides a method of inducing an immune response in an organism to a first virus, the method comprising the step of administering to the organism an immunogenic composition comprising an immunogen derivable from a second virus, wherein the immunogen derivable from the second virus has from 30% and 90% sequence identity to an equivalent immunogen derivable from the first virus.

Preferably the immunogen is derived from the second virus. The first virus and the second virus may be from the same family of viruses. The first virus and the second virus may be different species of virus.

The first virus and the second virus may be RNA viruses. The first virus and the second virus may be retroviruses. The first virus may be an HIV- 1 virus and the second virus may be an SIV virus or an HIV-2 virus. The first virus may be an HIV- 1 virus and the second virus may be an SIV virus, for example SIVmac239, SIVsmm, SIVcpz, SIVsme543 or SIVmac25 1. The immunogen may be a protein from the outer envelope of the virus. The immunogen may be an envelope protein from HIV, for example a gp l 60 protein or an immunogenic part thereof. The immunogen may be part of an envelope protein of HIV, for example a mature form of an envelope protein of HIV. The immunogen may be a gp l 40 protein of HIV. The immunogen may be a loop of a gp l40 protein, for example, the variable loops of an HIV- 1 gp l40 protein are highlighted in Figure 16. The equivalent loop regions from HIV-2 gp 140 or SIV gp l40 may be antigens derivable from the second virus.

The immunogenic composition may be for use as a vaccine

Surprisingly it was found that administration of an equivalent immunogen from a closely related, but not identical, virus can provide an immune response against the target virus. If a first virus is identified the skilled person may select an immunogen component of the first virus, for example part of the envelope protein. If a second related virus is found to have a similar equivalent protein that protein may be an immunogen derivable from a second virus and may be used in an immunogenic composition to raise antibodies against the first virus.

The second virus may be in the same family of viruses as the first virus but may be a different species of virus. An equivalent immunogen may be identified by aligning the polypeptide sequence of the immunogen from the first virus with polypeptide sequences from the second virus. An immunogen from the second virus may be chosen because it has between 30 % and 90% or more amino acid sequence identity to the antigen, or an equivalent immunogen, from the first virus, preferably between 40% and 90%, 50% and 90%, 60% and 90%, or 70% and 90% amino acid sequence identity to the antigen, or an equivalent immunogen, from the first virus.

The first virus and the second virus each encode at least one protein that is equivalent in each of the first and the second viruses and the sequences of these proteins can be aligned with 30% to 90% sequence identity.

The immunogen from the second virus may have between 30% and 90% protein sequence identity to the immunogen from the first virus. The immunogen from the second virus may have between 40% and 80% protein sequence identity to the immunogen from the first virus.

The immunogen derivable from the second virus may be gp l40 from SIVmac239 which has 36.6 % protein sequence identity with the gp l40 polypeptide sequence from HIV- 1 BX08 within the 666 amino acid overlap.

The immunogen derivable from the second virus may be gp l40 from HIV-2 which has 36. 1 % protein sequence identity with the gp l40 polypeptide sequence from HIV- 1 within the 671 amino acid overlap. The immunogen may be a whole protein or an oligomer, for example a homodimer, dimer or a trimer of proteins encoded by a virus. The immunogen may be a mature version of a protein that is truncated during cellular processing.

The immunogen may be a whole Env protein gp l 60 protein, gp l 40 protein, gp l 40 trimer, part of a gp l40 or a particular loop of a gp l40 or an immunogenic part thereof. The immunogen may comprise or consist of a trimer of three gp l40 proteins from an SIV virus, or an immunogenic part thereof, for example SIVmac239. The immunogen may comprise or consist of a trimer of three gp l40 proteins from an HIV-2 virus, or an immunogenic part thereof. The immunogen may comprise or consist of a mixed trimer comprising two or three different gp l40 proteins, or an immunogenic part thereof, for example a mixed trimer comprising at least one gp l 40 polypeptide from an SIV virus and at least one gp l40 polypeptide from an HIV-2 virus.

The immunogen from the second virus may be able to induce neutralising antibodies that bind to the equivalent immunogen from the first virus.

The first virus may be an HIV- 1 virus and the second virus may be an SIV virus or an HIV-2 virus. The first virus may be an HIV-2 virus and the second virus may be an SIV virus or an HIV- 1 virus.

Advantageously HIV- 1 , HIV-2 and SIV are related viruses but different viral species. It is therefore advantageous to use an immunogen protein from one of these viruses to produce neutralising antibodies that bind to the equivalent protein from one of the others in this group. The immunogen derivable from the second virus may be a gp l40 protein or a polynucleotide capable of expressing a gp l40 protein, or an immunogenic part thereof. The immunogen derivable from the second virus may be a gp l40 trimeric protein or a polynucleotide capable of expressing a gp l40 protein, or an immunogenic part thereof.

The immunogen derivable from the second virus may be a gp l40 trimeric Env protein from SIVmac239 or HIV-2 or a polynucleotide capable of expressing a gp l40 trimeric Env protein from SIVmac239 or HIV-2, or an immunogenic part thereof.

The immunogen derivable from the second virus may be a) a protein having the amino acid sequence set out in SEQ ID NO: 4 or SEQ ID NO: 6; b) a protein having greater than 99%, greater than 98%, greater than 95%, greater than 90%, greater than 85%, greater than 80% or greater than 75% sequence identity to a); or a polynucleotide capable of expressing a protein of a) or b). Variations in percent identity may be due, for example, to amino acid substitutions, insertions or deletions. Amino acid substitutions may be conservative in nature, in that the substituted amino acid has similar structural and/or chemical properties, for example the substitution of leucine with isoleucine is a conservative substitution.

In a further aspect the present invention provides a method of inducing an immune reaction wherein the method according to the first aspect of the present invention is a boosting step, and wherein the method further comprises a priming step of administering to the organism a priming composition comprising an immunogen derivable from the first virus, and wherein the priming step is performed before the boosting step.

In a further aspect the present invention provides a method of inducing an immune reaction wherein the method according to the first aspect of the present invention is a priming step and wherein the method further comprises a boosting step of administering to the organism a boosting composition comprising an immunogen derivable from the first virus, and wherein the priming step is performed before the boosting step.

The present invention provides a method of inducing an immune reaction comprising a priming step wherein an immunogen derivable from HIV- 1 is administered to an organism and a boosting step wherein an immunogen derivable from an SIV virus or an HIV-2 virus is administered to the organism, wherein the priming step is performed before the boosting step.

The present invention provides a method comprising a priming step wherein an immunogen derivable from HIV- 1 is administered to an organism and a boosting step wherein an immunogen derivable from an SIV virus or an HIV-2 virus is administered to the organism, wherein the priming step is performed before the boosting step.

The interval between the priming step and the boosting step may be at least about 1 , 2, 3, 4, 5, 6, 7, 8 or more weeks.

The priming step may be repeated at least 1 , 2, 3, 4, 5, 6, 7, 8 or more than 8 times.

The boosting step may be repeated at least 1 , 2, 3, 4, 5, 6, 7, 8 or more than 8 times.

The first virus may be HIV- 1 , and the immunogen derivable from the first virus may a mixture of 2-7 different env genes; and the second virus may be SIVmac239 and the immunogen derivable from the second virus may be a SIVmac239 gp l40 trimer, or an immunogenic part thereof.

The first virus may be HIV- 1 , and the immunogen derivable from the first virus may be a mixture of 3 different env genes; and the second virus may be SIVmac239 and the immunogen derivable from the second virus may be a SIVmac239 gp l40 trimer, or an immunogenic part thereof.

If the immunogen is an env gene it may be from a B-clade HIV- 1 strain, for example BaL JRFL, JRCSF HXB2, SF 162 MN.3, SC22.3C2 or RHP A.

An advantage of the present invention is that an immunogenic composition comprising an immunogen from a target or first virus is used to raise antibodies, and then the immune response is boosted by administration of an immunogenic composition comprising an equivalent immunogen from a second, preferably related virus, is that the result is an immune response focussed onto subdominant conserved elements of the immunogens.

The immunogenic composition comprising an immunogen derivable from the first virus may be administered before, after or simultaneously with the composition comprising an immunogen derivable from the second virus.

The immunogenic composition comprising an immunogen derivable from the first virus may preferably be administered before the composition comprising an immunogen derivable from the second virus.

The immunogen derivable from the first virus may be an Env protein or polynucleotide capable of expressing an Env protein, or an immunogenic part thereof.

In an embodiment the first virus is HIV- 1 , and the first immunogen is a mixture of 2-7 different env genes, and the second virus is SIVmac239 and the second immunogen is a SIVmac239 gp l40 trimer. In another embodiment the first virus is HIV- 1 , and the first immunogen is a mixture of three different env genes, and the second virus is SIVmac239 and the second immunogen is a SIVmac239 gp l40 trimer. In a further embodiment the first virus is HIV- 1, and the first immunogen is a mixture of three different env genes from clade B, and the second virus is SIVmac239 and the second immunogen is a SIVmac239 gp l40 trimer. In a further aspect the present invention provides a composition for use in inducing an immune response to a first virus, wherein the composition comprises an immunogen derivable from a second virus and wherein the immunogen derivable from the second virus has between 30% and 90% protein sequence identity to an equivalent immunogen derivable from the first virus.

The composition may be a boosting composition for use after a priming composition comprising an immunogen derivable from the first virus.

The composition may be a priming composition for use before a boosting composition comprising an immunogen derivable from the first virus.

The present invention provides use of a composition in the manufacture of a medicament for inducing an immune response to a first virus, wherein the composition comprises an immunogen derivable from a second virus and wherein the immunogen derivable from the second virus has between 30% and 90% sequence identity to an equivalent immunogen derivable from the first virus.

The immunogen derivable from the second virus may have between 30% and 80% sequence identity to the immunogen derivable from the first virus.

The immune response may be a neutralising antibody response. The immunogen derivable from the first virus may be an HIV-1 virus and the second virus may be an SIV virus or an HIV-2 virus.

The first virus may be an HIV-2 virus and the second virus may be an SIV virus or an HIV-1 virus.

The immunogen derivable from the first virus may be an Env gp l40 protein or a polynucleotide capable of expressing a gp l40 protein; and the immunogen derivable from the second virus may be an Env gp l40 protein or a polynucleotide capable of expressing a gp l40 protein.

The immunogen derivable from the second virus may be a gp l40 trimeric Env protein or a polynucleotide capable of expressing a gp l40 protein. The immunogen derivable from the second virus may be a gp l40 trimeric Env protein from SIVmac239 or HIV-2 or a polynucleotide capable of expressing a gp l40 trimeric Env protein from SIVmac239 or HIV-2.

The immunogen derivable from the second virus may be:

a) a protein having the amino acid sequence set out in SEQ ID 4 or SEQ ID 6; b) a protein having the amino acid sequence of amino acids 30 to 676 of SEQ ID NO 3 or of amino acids 31 to 677 of SEQ ID NO. 5;

c) a protein having 70%, 80%, 85%, 80%, 90%, 95%, 98% or more sequence identity to a) or b); or

c) a polynucleotide capable of expressing a protein of a), b) or c).

The first virus envelope protein may have an amino acid sequence as set out in SEQ ID NO. 1 or the first virus gp l40 has an amino acid sequence as set out in amino acids 30 to 655 of SEQ ID NO. 1 or SEQ ID NO. 2.

The composition may be formulated for simultaneous or sequential administration with a further composition comprising an immunogen derivable from the first virus. The immunogenic composition comprising an immunogen derivable from the first virus may be administered before, after or simultaneously with the composition comprising an immunogen derivable from the second virus. The immunogen derivable from the first virus may be an Env protein or polynuclotide capable of expressing an Env protein.

The first virus may be an HIV- 1 virus, the immunogen derivable from the first virus may be a mixture of three different env genes from clade B, and the second virus may be SIVmac239 and the immunogen derivable from the second virus may be a SIVmac239 gp l40 trimer.

A composition of the present invention may be used with an anti-HIV- 1 T-cell immunogen.

In a further aspect the present invention provides an immunogenic composition comprising an immunogen derivable from an SIV virus, wherein the composition is capable of eliciting an immune response against HIV- 1 , when administered to a human or non-human animal.

In a further aspect the present invention provides an anti-HIV- 1 or anti-HIV-2 antibody produced by a method of the present invention.

The immunogenic composition may comprise an immunogen derivable from an HIV- 1 virus and a) an immunogen derivable from an SIV virus; and/or an immunogen derivable from an HIV-2 virus, for raising an immune response against HIV- 1 virus.

In a further aspect the present invention may provide a kit comprising (i) a priming composition comprising an immunogen derivable from an HIV- 1 virus; (ii) a boosting composition comprising either or both of; an immunogen derivable from an SIV virus and an immunogen derivable from an HIV-2 virus, and (iii) instructions to administer the priming composition and the boosting composition to an organism. The kit may further comprise at point (iii) instructions to administer the priming composition and boosting composition with an interval of least one week. In step (iii) the interval may be at least 2, 3, 4, 5, 6, 7, 8 or more weeks.

In a further aspect the present invention may provide the use of a composition comprising an immunogen derivable from an SIV virus and/or a composition comprising an immunogen derivable from an HIV-2 virus for raising an immune response against HIV- 1.

In all aspects of the invention, the immunogen derivable from a first virus and/or the immunogen derivable from the second vector may be encoded by an adenoviral vector and/or a pox viral vector.

In the method, use or kit of the present invention the immunogens may be administered with an adjuvant.

In the method, use or kit according to the present invention the immunogens may be co-administered with another immunogenic composition or vaccine.

Examples of adjuvants may be compositions containing TLR agonsists or agonists to other pattern recognition receptors including for example C-type lectins, nucleotide-binding oligomerization domain (NOD)-like receptors, RIG-I, MDA-5 or danger associated molecular patterns (DAMP) (Heat shock proteins, uric acid, HMGB- 1 etc), aluminium salts, lipid compositions, saponins, squalene or derivatives thereof, or cytokines such as for example but not exclusively GM- CSF, IL- 15, IL-7. In a further aspect the present invention provides a method of vaccination against a first virus comprising the step of administering a priming composition comprising an immunogen derivable from a first virus and comprising a step of administering a boosting composition comprising an immunogen derivable from a second virus, wherein the immunogen derivable from the first virus has between about 30% and 80% sequence identity with the immunogen derivable from the second virus.

The first virus may be an HIV- 1 virus and the second virus may be an SIV virus or an HIV-2 virus.

In another aspect the present invention provides a composition comprising gp l40 from an SIV virus for use as an immunogenic composition or a vaccine. The composition, method or use of the present invention may elicit/produce a protective immune response when administered to a subject. The protective immune response may be against HIV- 1 virus.

The composition, method or use may be intended to produce a prophylactic or a therapeutic response. The composition, method or use may produce a prophylactic or a therapeutic response directed against HIV- 1 virus or HIV-2 virus. The composition, method or use may produce a prophylactic vaccine against HIV- 1 or HIV-2. The composition, method or use may be capable of producing a protective immune response to HIV- 1 or HIV-2.

The phrase "producing a protective immune response" as used herein means that the composition is capable of generating a protective response in a host organism, such as a human or a non-human mammal, to whom it is administered. Preferably a protective immune response protects against subsequent infection by HIV- 1 virus and/or HIV-2 virus

Suitable acceptable excipients and carriers will be well known to those skilled in the art. These may include solid or liquid carriers. Suitable liquid carriers include water and saline . The proteins of the composition may be formulated into an emulsion or they may be formulated into biodegradable microspheres or liposomes. Suitable adjuvants will be well known to those skilled in the art, and may include Freund's Incomplete Adjuvant (for use in animals), and metal salts, such as aluminium or calcium salts. The composition may also comprise polymers or other agents to control the consistency of the composition, and/or to control the release of the immunogen from the composition.

The composition may also comprise other agents such as diluents, which may include water, saline, glycerol or other suitable alcohols etc; wetting or emulsifying agents; buffering agents; thickening agents for example cellulose or cellulose derivatives; preservatives; detergents, antimicrobial agents; and the like.

Preferably the active ingredients in the composition are greater than 50% pure, usually greater than 80% pure, often greater than 90% pure and more preferably greater than 95%, 98% or 99% pure. With active ingredients approaching 100% pure, for example about 99.5% pure or about 99.9% pure, being used most often.

The composition of the present invention may be used as vaccine against infections caused by HIV- 1 virus and/or HIV-2 virus. The composition may be used as a vaccine directed to HIV- 1 and/or HIV-2. The vaccine may be administered prophylactically to those at risk of exposure to HIV- 1 and/or HIV-2 and/or therapeutically to persons who have already been exposed to HIV- 1 and/or HIV-2.

Preferably, if the composition is used as a vaccine, the composition comprises an immunologically effective amount of immunogen/antigen. An "immunologically effective amount" of an immunogen is an amount that when administered to an individual, either in a single dose or in a series of doses, is effective for treatment or prevention of infection by HIV- 1 and/or HIV-2. This amount will vary depending upon the health and physical condition of the individual to be treated and on the antigen. Determination of an effective amount of an immunogenic or vaccine composition for administration to an organism is well within the capabilities of those skilled in the art. A composition according to the invention may be for oral, systemic, parenteral, topical, mucosal, intramuscular, intravenous, intraperitoneal, intradermal, subcutaneous, intranasal, intravaginal, intrarectal, transdermal, sublingual, inhalation or aerosol administration.

The composition may be arranged to be administered as a single dose or as part of a multiple dose schedule. Multiple doses may be administered as a primary immunisation followed by one or more booster immunisations. Suitable timings between priming and boosting immunisations can be routinely determined.

A composition according to the invention may be used in isolation, or it may be combined with one or more other immunogenic or vaccine compositions, and/or with one or more other therapeutic regimes.

The skilled person will appreciate that any of the preferable features discussed above can be applied to any of the aspects or embodiments of the invention.

Preferred embodiments of the present invention will now be described, merely by way of example, with reference to the following figures and examples.

Figures 1A and IB - show trimerization of SIV_MAC239 gp l40 and binding to human CD4. A) Non-reducing (N/R) and reducing (Red) SDS PAGE analysis of SIVmac239 and HIV- 1YU2 gp l40s. The SIV_MAC239 gp l40 protein runs as a high molecular weight band under non-reducing conditions indicating that the three polypeptides of the trimer are covalently linked by disulfide bonds between the three gp l40 chains. HIV- 1_YU2 gp l40 runs as a smear under non-reducing conditions suggesting that the three polypeptides of the trimer are not covalently linked. B) Surface plasmon resonance analysis of SIV_MAC239 gp l40 and HIV- 1_UG₃₇ gp l40 binding to human CD4 and anti-HIV- 1 NAbs. SIV_MAC239 (right panel) or HIV- 1_UG₃₇ (left panel) were immobilized and their binding to tetrameric soluble human CD4 (CD4-IgG2) as well as mAb F 105, HGP68 and HR10 was measured. Figure 1C - shows surface plasmon resonance analysis of mAb and soluble CD4 binding to SIVmac239. (A) Interaction of an extended panel of mAb with HIV- 1 UG37 or SIVmac239 is shown as in Fig. I B. (B) Kinetic data of sCD4 interaction with HIV- 1 and SIV gp l40s was recorded and fitted to a 1 : 1 Langmuir binding model. Traces are plotted in black with curves showing the fit results overlain in red (pale grey) .

Figure 2 - shows a schematic representation of the immunization regime . A) Schematic immunization schedules of two independent studies (n= 48) with arrows depicting immunizations. Sera were collected before immunization (week 0) . Animals (New Zealand white rabbits) received a mix of three clade B env-DNA plasmids at weeks 0 (twice first week), 4, 8 and 12 (200 μg DNA by i.d. electroporation). Sera were collected 4 weeks after last DNA immunization (w l 6) . Animals then received a heterologous protein trimer SIV_mac239 by i.d. injection and sera were collected 4 weeks after first protein boost (50 μg) (w20). Animals received a second protein boost with SIV_mac239 gp l40 trimer 4 weeks after the first protein immunization and sera were collected 2 weeks after second protein boost (50 μg) (w22). Three months later sera were analyzed for memory responses (w34). In the first study, twelve animals were included in Gr. 1 and the second study entailed six animals in this group. In the first study, Gr. 2 (n= 12) received AAC adjuvant during the DNA priming phase and in the second study Gr. 2 (n=6) received a TLR5 agonist pFliC. Gr. 3 (n=6) received control DNA and albumin protein and Gr. 4 received three immunizations with protein trimer SIVmac239 by i.d. injection (n=6).

Figure 3 - shows induction of high-titre neutralizing antibodies against tier 1 and tier 2 clade B viruses through the highly heterologous DNA- prime protein-boost regime . Data are shown for individual animals from

Groups 1 and 2 combined (n=24) (A-I) . Lines represent median. Sera were collected before immunization (wO), 4 weeks after last DNA immunization (w l 6), 4 weeks after first protein boost (w20) and 2 weeks after second protein boost (w22) . Three months later sera were analyzed for the longevity of responses (w34). High ID₅₀ neutralizing titres were detected in a TZM-bl assay against tier 1 SF 162 (A) and MN.3 (B). Purified IgGs were tested in a TZM-bl assay against SF 162 and BX08 (C and D). Percent neutralization is shown using 130 μg/ml IgG final concentration approximately corresponding to a 1/80 serum dilution and dotted line indicates ID₅₀. Endpoint neutralization titres (ID₅₀) in sera were tested in A3R5.7 assay against clade B viruses 9020 (E) RHP A (F) and SC22.3C2 (G) . Endpoint neutralization titres (ID₅₀) in sera were tested in TZM-bl assay against clade C (MW965.26) (H) and clade AE (TH023.6) (I) viruses. Representative examples (data from MN.3) of HIV- 1 neutralization results obtained in animals that received control DNA and albumin (Gr.3) or only SIV_MAC239 protein without a DNA prime (Gr.4) (J) as well as SIVmac25 1 neutralization (K) were determined in the TZM-bl assay. Significant values are indicated by * * * p<0.001 , * *p<0.01 and *p<0.05 using One-Way ANOVA, Kruskal-Wallis with Dunns s Multiple

Comparison Test.

Figure 4 - shows epitope mapping which reveals preferential binding to C I , C2, V2, V3 and V5. (A) Selected sera (n=2 per group from the first study) from time points after HIV- 1 DNA-prime (w l 6) and after the first

SIV_MAC239 protein boost (w20) were subjected to epitope mapping by pepscan peptide arrays using heterologous HIV- 1C_N54 peptides. (B) The change in response intensity after SIV_MAC239 protein boosting is shown for peptides that were undetectable after DNA immunizations (white) or showed positive responses after DNA priming (grey) (C) The percentage of positive peptides in the peptide array is shown for week 16 and week 20. (D) ELISA binding titres to indicated peptides from HIV- 1 or SIV_MAC239 were determined after the second protein boost (w22) in animals immunized without (Gr. 1 ; n=6) or with a cellular adjuvant AAC during the priming phase (Gr. 2; n=6) (mean+SEM). Significant values are indicated by *p<0.05, * *p<0.01 and * * * *p<0.0001 as well as ns=not significant using a Mann-Whitney test (B), a paired t test (C), or a oneway ANOVA of log-transformed data with a Bonferroni's Multiple Comparison Test (D). Figure 5 - shows neutralization against the SF 162 virus is targeted to the V3 region in the majority of animals. IgG from animals from the first study were assessed for their capacity to neutralize HIV- 1 S_{F I62} in the TZM-bl assay in the presence of blocking peptides and representative individual results are displayed. IgG were purified from sera obtained at the week 22 time point two weeks after the second protein boost. Percent neutralization is depicted on the y-axis and concentration of purified IgG on the x-axis. (A) Neutralization is displayed for three representative individual animals in the absence (black) or presence of a peptide covering the V3 region from HIV- 1 clade B virus MN3 (dark grey) or a scrambled control peptide (light grey). Peptide controls without IgG did not result in significant enhancement or neutralization of the virus (Fig.8) . (B) Pooled analysis of the neutralization (no peptide, white) and inhibition by a short V3 peptide from clade A containing the GPGQ motif (light grey) or clade B containing the GPGR motif (dark grey), respectively. Results from peptide inhibition without IgG as well as control neutralization using pre-immunization IgG in combination with the peptides (wO) did not show significant enhancement or neutralization of the virus. Significant values are indicated by * * * p<0.001 , * *p<0.01 and

*p<0.05 as well as ns=not significant using a two-way repeated measures ANOVA with a Bonferroni's Multiple Comparison Test.

Figure 6 - shows potent neutralization ID50 titres after immunization. Serum ID50 titres obtained from individual animals at week 22 from the first study (Group (Gr) l (no adjuvant) and 2(AAC adjuvant)) are coded as follows: * * * * *boxes, titre<20; * * * *boxes, titre< 100; * boxes, titre<500; * * *boxes, titre< 1000; and * *boxes, titre > 1000. Grey boxes, not determined. Additional viruses tested in TZM-bl assay and all samples below 100; JR-FL, Q23. 17, 6535.3, QH0692.42

Figure 7 - shows high titre binding antibodies elicited by SIV_MAC239 gp l40 protein boost following HIV env prime. (A) Endpoint titres (log l O) of binding to SIV_MAC239 gp l40 (white bars) or heterologous HIV- 1_UG₃₇ gp l40 (black bars) as measured by ELISA analyzing sera obtained before immunizations (week 0) and after immunizations at w22 from the first study. Data are depicted as median and range. (B) and (C) show endpoint titres of binding to SIV_MAC239 gp l40 and heterologous HIV- 1_UG₃₇ gp l40, respectively, in sera obtained at weeks 0 (black bars), 16 (grey bars) and 20 (white bars) from the second study. Statistically significant lower endpoint titres against SIV_MAC239 were found in Gr. 3 and against HIV- 1_UG₃₇ in Groups 3 and 4 when compared with Groups 1 and 2 (One-way ANOVA using Bonferroni's Multiple Comparison Test p<0.05) .

Figure 8 - shows an entire panel of animals tested in the peptide inhibition assay. Individual results of the peptide inhibition assay as presented in Fig.5A. Percent neutralization is depicted on the y-axis and concentration of purified IgG on the x-axis. (A) Neutralization is displayed for individual animals in the absence (black) or presence of a peptide covering the V3 region from HIV- 1 clade B virus MN3 (dark grey) or a scrambled control peptide (light grey) . Peptide controls without IgG did not result in significant enhancement or neutralization of the virus.

Figure 9 - shows SEQ ID NO. 1 which is the sequence of HIV- 1 (BX08) gp l 60. SEQ ID NO. 2 the sequence of gp l40 is highlighted.

Figure 10 - shows SEQ ID NO. 3 which is the sequence of HIV-2 409. 14 gp 160. SEQ ID NO. 4 the sequence of gp l40 is highlighted. The gp l40 protein may be used as an immunogen.

Figure 11 - shows SEQ ID NO. 5 which is the sequence of SIVmac239 gp l 60. SEQ ID NO. 6, the sequence of gp l40 is highlighted. The gp l40 protein may be used as an immunogen.

Figure 12 - shows a sequence alignment of the whole Env protein from HIV-2, SIVmac239 and HIV- 1 (BX08). Figure 13 - shows a sequence alignment of the mature gp l40 protein immunogens from HIV-2 and SIVmac239.

Figure 14 - shows a sequence alignment of the mature gp l40 protein immunogens from HIV- 1 (BX08) and SIVmac239.

Figure 15 - shows a sequence alignment of the mature gp l40 protein immunogens from HIV- 1 (BX08) and HIV-2. Figure 16 - shows the sequence of HIV- 1 BX08 gp l60 with the leader peptide, variable loops V I to V5 and the membrane spanning domain highlighted.

A HIV- 1 vaccine capable of eliciting broadly neutralizing antibodies has been elusive for many years, yet is crucial to combat the global AIDS pandemic. Whilst a single viral envelope immunogen can elicit neutralizing antibody responses to a narrow range of viral strains, the results reported here demonstrate an immunization strategy composed of a trivalent HIV- 1 envelope DNA prime, followed by a SIV_mac239 envelope protein boost that focuses the immune response to the most conserved parts of the Env and SIV envelope proteins. Neutralizing antibodies to HIV- 1 were elicited by the HIV- 1 Env DNA prime and were significantly boosted by the use of the SIV_mac239 p l40 trimer protein boost. Mapping of the responses revealed that the elicited antibodies were specific to multiple conserved elements on the HIV- 1 envelope . This strategy demonstrates that a highly divergent prime-boost regimen can be exploited for HIV- 1 vaccine development.

The immunological principle put forward here is based on sequential host exposure to divergent Envs in order to favor the induction of immune responses against other regions than the highly immunogenic variable epitopes. To achieve this several heterologous immunizations of rabbits were used, consisting of DNA vaccination with multiple HIV- 1 Env constructs followed by boosting with recombinant trimeric Env protein. As a novel strategy to focus antibody responses primarily towards conserved epitopes, boosting was based on a highly heterologous trimeric Env protein derived from SIV_mac239. Indeed HIV-1 and SIV_mac239 gp l40 proteins have only about 30% sequence identity but nevertheless bind human CD4 and display significantly conserved topological architecture. Additionally, the higher stability of SIV_mac239 trimers when compared to those generally produced from HIV-1 Env is likely to provide additional advantages during immunization. In conclusion, the vaccination strategy described in this study induced high titre heterologous NAbs against both tier 1 and 2 viruses within clade B as well as cross-clade activity against clade AE and C viruses, including HIV-1 Env-specific responses to conserved epitopes primarily in the C 1,C2, V2, V3 and V5 regions.

RESULTS

Production of stable covalent SIV_mac239 gpl40 for heterologous boost

DNA env clade B gp l40 was delivered as a mixture composed of three different, synthetic, codon optimized envs (from the clinical isolates HIV-1 Bx08, ctl21 and ctl27) to increase the proportional concentration of epitopes derived from shared regions. Boosting with the highly heterologous trimeric SIV_mac239 gp l40 was used to enhance antibody titres to regions involved in viral entry, which are conserved between priming and boosting antigens. All gp l40 trimers were produced in HEK 293T cells using transient expression plasmid DNA transfection. SIV_mac239 gp l40 proteins displayed enhanced trimerization compared to HIV-1 gp l40, as demonstrated by SDS PAGE analysis (Fig. lA). While the SIV_mac239 gp l40 protein migrated as a tight high molecular weight band on non-reducing SDS PAGE, suggesting that the three gp l40 chains are covalently linked, the HIV-1 gp l40 bands were less defined, indicating weakly-associated trimers and the presence of dimeric and monomeric species. Both gp l40 trimers migrated as single species under reducing conditions, confirming the presence of inter-chain disulfide bonds and the integrity of the monomers. Surface plasmon resonance binding analysis revealed that SIV_mac239 gp l40 binds to soluble recombinant human CD4. However, no binding to SIV_mac239 gp l40 was detected using a panel of mAb against HIV-1 gp l60 including F105, HGP68 and HR10 which recognize the CD4 binding site, the V2 and the V3 regions, respectively (Fig. IB and Fig. lC). Potent and broad neutralization elicited by the heterologous prime boost strategy

To mimic the long-term antigenic exposure required for the development of bNAbs in HIV- 1 -infected individuals, an intense immunization regimen was adopted, which consisted of repeated DNA immunizations of rabbits over a four month period prior to boosting with SIV_mac239 gp l40 trimeric Env. Plasmid DNA was delivered by intradermal (i.d.) injections in the first week (day 0 and 2) as well as at weeks 4, 8 and 12 (Fig.2). Each i.d. injection was followed by electroporation since it was demonstrated to substantially increase immunogenicity in both animal and human studies. Two SIV_mac239 gp l40 boosts (50 μg/animal/time-point) were delivered i.d. at weeks 16 and 20 without adjuvant in both groups 1 and 2. Additionally, it was evaluated if an adjuvant, consisting of activated apoptotic cells (AAC) that engage TLR4, would increase DNA priming efficiency (Gr. 2) (Fig.2). Subsequently, a second independent study was performed in which group 2 received a TLR5 agonist that was delivered as a DNA plasmid (pFliC) together with the DNA prime (Gr. 2) (Fig.2). Furthermore, it included a negative control group receiving empty plasmid and albumin (Gr. 3) and a group, which received only the recombinant SIV_mac239 trimer (Gr. 4) to address if neutralization responses against HIV- 1 could be induced without HIV- 1 DNA env priming.

Sera were analyzed for neutralizing activity against a panel of tier 1 viruses in the TZM-bl assay as previously defined by Seaman et al. (2010) J Virol 84: 1439- 1452, and responses are displayed as 50% inhibitory dose (ID₅₀) titres (Fig.3A). Neutralization responses from Groups 1 and 2 of the first study are displayed together as no significant differences in NAb activity were found in the group receiving the AAC adjuvant (Figure 6). A significant induction of neutralizing activity was measured against heterologous SF 162 and MN viruses following the DNA priming phase (w l 6) with strongly increased neutralization titres after the first protein boost (w20, Fig.3A-B). Measurements with purified serum IgG confirmed the induction of neutralization activity as well as the strong effect of the SIV_mac239 boost (Fig.3C-D). Furthermore, significant neutralization titres were measured against tier 1 and 2 viruses using the A3R5.7 assay (Fig.3E-G) as well as against HIV-2 (Suppl. Table 1). Interestingly, the second protein boost delivered four weeks after the first boost did not always significantly increase the neutralization titres. Nevertheless, when long-term persistence of neutralization was evaluated three months after the final SIV_mac239 protein boost (w34), responses were detected against both tier 1 (Fig.3A, B and E) and tier 2 clade B viruses (Fig.3F-G). Furthermore, significant cross-clade neutralizing activity was also detected against tier 1 viruses from clade C (MW965.26) and CRF01 -AE (TH023.6) (Fig.3H-I). In line with the measured antibody binding titres (Fig.2C), SIV_mac239 gp l40 boosting without prior priming (Gr.4) did not induce NAbs against HIV- 1 viruses (Fig.3J), whereas significant responses against SIV_mac25 i were observed (Fig.3K).

Taken together these data demonstrate the induction of high titres of NAb activity against both tier 1 and tier 2 clade B viruses with evidence of cross-clade NAb activity as well as durable antibody responses that lasted for at least three months after the final immunization. Overall, a more potent reactivity was observed against heterologous virus from clade B compared to clades AE or C, likely caused by the use of clade B envs during the priming phase .

Epitope mapping reveals preferential antibody binding to CI , C2, V2, V3 and V5

The prevalence of Env-specific binding IgGs in serum against both SIV_mac239 gp l40 and heterologous clade A HIV- 1_UG37 gp l40 was assessed by ELISA. High binding antibody titres against both antigens were detected in all animals following the protein boost (w22), without any demonstrated effect of the AAC adjuvant (Fig.7A). The second study confirmed the induction of high binding antibody titres against both SIV_mac239 and HIV- 1_UG37 gp l40. High ELISA titres were detected after DNA immunizations already before the first protein boost (w l 6) and found to be generally increased by the protein boost (Fig.7B,C). Similarly to the results obtained with ACC, no difference in binding titres was detected between the groups that did or did not receive the pFliC adjuvant during the DNA priming phase (Fig.7B,C, Gr.2). The empty vector and albumin protein control group did not mount any SIV_mac239 or HIV- 1 _UG37 gp l40-binding antibodies (Fig.7B,C Gr.3). Importantly, repeated administration of SIV_mac239 trimer without HIV- 1 DNA priming did not elicit high titre binding antibodies to HIV- 1_UG37 gp l40 even though responses to SIV_MAC239 were induced (Fig.7B,C, Gr. 4). Conversely, binding responses towards SIV_MAC239 were induced already by the HIV- 1 DNA prime prior to the SIV_MAC239 Env boost (Fig.7B, Gr. 1 and 2). Epitope mapping using linear and circularized 15-mer HIV- 1C_N54 peptides was performed to obtain additional insights into the specificities of the antibody binding response . Sera from week 16 primarily recognized epitopes in the C I and V3 regions, and to a lesser extent in the V2, C2 and V5 regions (Fig.4A). Notably, almost all the observed antibody responses increased following the SIV_MAC239 gp l40 boost (Fig.4A-C). Indeed, antibody-binding responses that were detectable already after priming (w l 6) displayed on average a significantly stronger increase after the boost (Fig. 4B). In addition, antibody binding to some peptides that were not recognized after DNA priming was detected after protein boost, demonstrating an increased breadth following the SIV_MAC239 boost (Fig.4B and C).

Peptides from regions in C I , V2, V3 and V5 were used in ELISA in order to assess the impact of the AAC adjuvant on binding titres against individual epitopes. Although the AAC adjuvant did not improve binding antibody titres against the complete gp l40 trimers, it significantly increased responses against all the HIV- 1 peptides tested (Fig.4D). Altogether, these results indicate that the SIV_MAC239 protein boosted the primed HIV- 1 responses, and led to broadening of the recognition, while the addition of AAC during the priming phase induced higher antibody binding titres to C I , V2, V3, and V5.

The neutralization of HIV- 1 S_{F I62} was assessed in the presence of a cyclic peptide that comprises the entire V3 region of MN HIV- 1 gp l20 (residues 266 to 301) in sera obtained following two protein boosts (w22) (Fig.5A, Fig. 8). The neutralization inhibition assay demonstrated recognition of the peptide in sera from all of the animals tested and blocking of a major part of the NAb responses in eight out of the twelve animals tested. Further neutralization assays were performed with shorter peptides that were localized centrally on the V3 -crown motifs of clade B (TRKSIYIGPGRAFHTT) or clade A (KSVHIGPGQAFYAT) (Fig.5B) . Although, a significant portion of NAb responses was inhibited by the clade B peptide with the GPGR motif, a significant heterologous inhibition by the clade A peptide was also detected despite its absence from the immunization regime (Fig.5B). CONCLUSION

The data presented herein demonstrates the induction of broad and potent HIV- 1 Env-specific NAbs against clade B tier 1 and tier 2 viruses as well as cross- reactivity against clade AE and clade C viruses through repeated DNA env mixture priming followed by a highly heterologous boost based on relatively stable SIV_mac239 gp l40 trimers.

High binding titres against HIV- 1 were measured (Fig.2B, D) and these were significantly increased by the SIV_mac239 boost (Fig.4). Furthermore, the data obtained suggest a broadening and higher magnitude ELISA end-point binding titres to peptides in C I , V2, V3 and V5 if an adjuvant triggering TLR4 (AAC) was used during the DNA priming phase. However, and most importantly, the boost resulted in a robust increase of the bNAb responses against HIV- 1. The results presented here highlight the potential of SIV_mac239 gp l40 trimers for the enhancement of potent HIV- 1 neutralization. Furthermore, the high neutralization titres observed in this study suggest that repeated DNA priming followed by boosting with a heterologous protein trimer represents a platform for vaccination.

In conclusion, the present study provides a framework for the induction of HIV- 1 Env specific broad and potent NAbs against clade B tier 1 and tier 2 viruses as well as cross-reactivity against clade AE and clade C viruses by repeated DNA env mixture priming followed by a very heterologous boost using a relatively stable trimeric SIV_mac239 protein. MATERIALS AND METHODS

Animals

New Zealand White (male and female) rabbits ( 10- 12 weeks of age at start of experiment, approximately 3 kg) were housed at the animal facility of the Swedish Institute for Infectious Control according to directives and guidelines of the Swedish Board of Agriculture and the Swedish Animal Protection Agency. The study was performed under approval of the Stockholm North Ethical Committee on Animal Experiments. Expression and purification of recombinant gpl40

SIV_MAC239, HIV- 1_UG₃₇ and _YU2 gp l40 were produced following transient transfection of 293T cells cultured in multilayer Cell Bind Hyperflasks (Corning) in high glucose DMEM (Sigma) supplemented with 10% FCS (Sigma) and Penicillin- Streptomycin solution (Sigma) . Two mg plasmid DNA was incubated with 3.6 mg PEI in media without FCS for 30 minutes to allow complex formation. This was added to cells and brought to 500 ml with DMEM containing 2% FCS. Supernatants were collected after 48 hours and fresh media, containing 10% FCS was added to the cells for a further 48 hours at which point the media was exchanged once again. All supernatant was centrifuged at 7000 x g for 4 hours to remove cell debris, and passed through a 0.22 μιη filter. After adjusting to pH 8 using 1 M Tris HC1 (Sigma), media was passed over a cobalt chloride metal- affinity column made from Talon superflow resin (Clontech).

After washing with 2 column volumes of 0.015 M Tris Buffered Saline (Sigma), protein was eluted with 250 mM imidazole. The eluted gp l40 was concentrated and separated by gel filtration chromatography using a Superdex200 26/60 size- exclusion column (GE Healthcare). Fractions corresponding to the trimer were identified and further purified using GNA-lectin (Vectorlabs) to specifically bind the glycoprotein. A further SEC fractionation allowed separation of pure gp l40.

Surface plasmon resonance

Surface plasmon resonance experiments were performed on a Biacore 3000 (Biacore Inc., Sweden) in HBS-EP buffer (Biacore Inc.) at 37°C. 700 RUs of gp l40 were immobilized on a CM5 sensor chip using standard amine coupling at pH 4.5. Binding responses were measured by injecting mAbs or CD4-IgG₂ over the surfaces for 5 min at 50 μΐ/min followed by a 5 min dissociation phase. For kinetic analysis a 2-fold dilution of sCD4 starting at 20 nM was used under similar conditions. Between cycles, the sensor surface was regenerated by two 30- second injections of 10 mM glycine, pH 2.0. Data were analyzed in the BIA evaluation software (V 4.0. 1). Double referencing was performed using a blank control flow cell as well as a buffer injection and kinetic data were fitted to a 1 : 1 Langmuir binding model. DNA vaccine plasmids

The construction of BX08 gp l40 (Genbank JX473289) plasmid used codons from highly expressed human genes as described earlier in Corbet et al. (2000)AIDS Res Hum Retroviruses 16: 1997-2008, Vinner et al. (2003) J Gen Virol 84: 203- 213 and Vinner et al. (2006) APMIS 1 14: 690-699, and two other primary Envs from Danish patients (ctl21 (JX473290) and ctl27 (JX473291) were similarly codon optimized and cloned in the same expression plasmid with the key elements CMV IE promotor-enhancer, tPA secretion signal, and a bovine growth hormone poly A signal. Clones were verified by sequencing. Immunizations

Groups of 6 to 8 rabbits, 9- 1 1 weeks old, were immunized at weeks 0 (twice first week), 4, 8 and 12 with a mix of three clade B env-DNA plasmids encoding HIV- 1 env gp l40 i.d. with the Derma Vax EP device (CytoPulse Sciences/Cellectis, Romainville, France) (Fig.2A). A total concentration of 200 μg DNA was injected i.d. at two injection sites, as described in Roos et al. (2009) PLoS One 4: e7226. Blood for collection of sera and PBMC was taken from the ear vein before immunizations (time point 0 (w0)) and 2 weeks after the last DNA immunization (w l 6) . One group of animals (n= 12) received a cellular adjuvant consisting of allogeneic activated cells (AAC), prepared as previously described Spetz et al. (2002) J Immunol 169: 5771 -5779. In short, allogeneic rabbit splenocytes were isolated after passage through a sterile mesh. Splenocytes (2xl 0⁶/ml) were cultured in medium containing RPMI 1640 (GIBCO Life Technologies, Paisley, United Kingdom), supplemented with 1 % HEPES (N-(2-hydroxyethyl)piperazine- N'-2-ethanesulfonic acid) (GIBCO), 2 mM 1-glutamine (GIBCO), 1 % streptomycin (GIBCO), and 1 % penicillin (GIBCO), 10% endotoxin-free fetal bovine serum (FBS) (GIBCO) and Concanavalin A (2,5 μg/ml) (Sigma) for 48 hours. Cells were harvested and frozen in 10% DMSO and 90% FBS. On the day of immunization cells were thawed, washed twice in PBS and exposed to 150 Gy gamma-irradiation for apoptosis induction, as previously described Spetz et al. ( 1999) J Immunol 163 : 736-742 and Bergsmedh et al. (2001) Proc Natl Acad Sci U S A 98 : 6407-641 1. The gamma-irradiated cells were diluted in PBS and mixed with the DNA before administration. In total each rabbit received 10x106 AAC per injection time point. All animals then received a heterologous protein trimer SIV_MAC239 (50 μg) by i.d. injection (no adjuvant) and sera was collected 4 weeks later (w20). A second identical boost was performed 4 weeks after the first protein immunization and blood was collected 2 weeks later (w22). Three months later blood samples were analyzed for memory responses (w34) . A second repeat study with 24 animals divided into 4 groups was performed: the first group (n=6) received the DNA-env clade B mix priming at weeks 0 (twice first week), 4, 8 and 12 delivered by electroporation; a second group received the addition of a TLR5 agonist encoding flagellin (pFliC) as previously described Applequist et al. (2005) J Immunol 175 : 3882-3891. The env DNA plasmids were mixed with 200 μg pFliC plasmid before administration by electroporation. These two groups then received a heterologous protein trimer SIV_MAC239 (50 μg) by i.d. injection (no adjuvant) and sera was collected 4 weeks later (w20). The third group received empty vector plasmids and albumin as protein boost. The fourth group received protein trimer SIV_MAC239 (50 μg) by i.d. injection at weeks 0, 8 and 16. Sera was separated and stored at -20°C until IgG purification or direct analysis.

Enzyme-linked immunosorbent assay (ELISA)

Endpoint binding titres were determined as described previously Wegmann et al. (201 1) PLoS One 6: e 15861. Briefly, serially diluted serum samples were added to microtitre plates coated with 0.5 μg/ml gp l40 (HIV- 1_UG₃₇ or SIV_MAC239) or HIV- 1C_N54 C I (74-CVPADPNPQEMVLEN, HXB2 numbering), HIV- 1 _CN54 V2 ( 175 - LFYRLDIVPLTKKNY), HIV- 1 _CN54 V3 (300-NNTRKSIRIGPGQTF), HIV- 1C_N54V5 (461 -EPNDTETFRPGGGDM), SIV_MAC239 V2 ( 169-

KFTMTGLKRDKTKEYNETWY) or SIV_mac239 V3 (3 19-

VLPVTIMSGLVFHSQPINDR) peptides and blocked with 2% bovine serum albumin in washing buffer (0.05 % Tween-20 in PBS). Bound antibodies were detected with anti-rabbit IgG-HRP (Sigma-Aldrich), developed with 1 -STEP Ultra TMB-ELISA substrate (Thermo Fisher Scientific) and OD values were read at 450 nm. Data were analyzed by subtracting the background followed by fitting of a sigmoidal dose-response curve to each dataset. Endpoint titres were defined as the concentration at which the curve reached the threshold (0.01), which was greater than two standard deviations above background. HIV peptides were obtained from the EU Eurovac consortium. Peptide Scanning

Overlapping linear 15-mer and circularized 15-mer peptides based on gp l40 of HIV- 1 C_N54 were tested for reactivity against heat-inactivated rabbit sera by Pepscan Therapeutics (Netherlands). Positive responses were defined as higher than two times the median of all peptides tested. The effect of boosting was determined by dividing the ELISA value after boosting by the ELISA value after DNA prime for each peptide. Peptide binding breadth for each animal was calculated as the percentage of peptides that showed positive responses. For graphical representation, average ELISA values of 9AA windows were calculated for each position.

Neutralization assays

IgG were purified from serum using Protein G HP SpinTrap columns (GE Healthcare) according to the manufacturer's instructions. Eluted IgG were quantified spectrophotometrically with yields ranging from 2 to 4 mg/ml. The purified IgG or sera were assayed in pseudovirus neutralization assays using TZM-bl, expressing CD4 as well as both CCR5 and CXCR4, conducted in triplicates as described previously http://www.europrise.org/neutnet sops.html; SOP 10) or using A3R5 cells. A3R5 cells [64] were obtained from Drs. Jerome Kim and Robert McLinden at the US Medical HIV Research Program (MHRP) . This is a human CD4⁺ lymphoblastoid cell line (CEM/A3.01) that was engineered at the US MHRP to express CCR5. Infectious molecular clones of HIV- 1 carrying the entire ectodomain of the virus of choice and a Tat-regulated LucR reporter gene were obtained from Drs. Christina Ochsenbauer and John Kappes at the University of Alabama, Birmingham. Briefly, a dose of virus that generates approximately 50,000 relative luminescence units (RLU) after 4 days of infection was incubated with serial 3 -fold dilutions of test sample in duplicate in a total volume of 150 μΐ for 1 hr at 37°C in 96-well flat-bottom culture plates. Exponentially dividing A3R5 cells (90,000 cells in 100 μΐ of growth medium containing 25 μg/ml DEAE dextran) were added to each well. One set of control wells received cells + virus (virus control) and another set received cells only (background control). A panel of pseudoviruses was used and prepared as previously described Montefiori DC (2009) Methods Mol Biol 485 : 395-405. Infection levels were determined after 48 h by measuring firefly luciferase activity in TZM-bl and after four days by measuring Renilla luciferase activity in A3R5.7 cells. The ID50 was calculated as the reciprocal plasma dilution causing 50% reduction of relative light units compared to the virus alone (without test sample). Peptide competition neutralization assays

Peptide inhibition of IgG neutralization was measured using a modified pseudovirus neutralization assay in which the purified IgGs were pre-incubated for 30 min with peptide dissolved in DMSO at a final concentration of 16 μg/ml prior to addition of the pseudovirus. The HIV-1 MN V3 peptide (CTRPNYNKRKRIHIGPGRAFYTTKNIIGTIRQAHC, EVA7019), The GPGR HIV-1 SF2 clade B V3 peptide (TRKSIYIGPGRAFHTT, ARP797), the GPGQ HIV-1 consensus clade A peptide (KSVHIGPGQAFYAT, ARP7012.1) and the scrambled control (ARP7099) were obtained from the EVA Centre for AIDS Reagents, NIBSC, UK.

Statistical analyses

Figures as well as statistical analyses were prepared using Graph Pad Prism statistical software Version 4.03 (GraphPad Software, La Jolla, CA) according to the statistical tests described in the figure legends.

Claims

1. A composition for use in inducing an immune response to a first virus, wherein the composition comprises an immunogen derivable from a second virus and wherein the immunogen derivable from the second virus has between 30% and 90% protein sequence identity to an equivalent immunogen derivable from the first virus.

2. A composition according to claim 1 wherein the immunogen derivable from the second virus has between 30% and 80% sequence identity to the immunogen derivable from the first virus.

3. A composition according to claim 1 or claim 2 wherein the immune response is a neutralising antibody response.

4. A composition according to any one of the preceding claims wherein the first virus is an HIV- 1 virus and the second virus is an SIV virus or an HIV-2 virus.

5. A composition according to any one of the preceding claims wherein the first virus is an HIV-2 virus and the second virus is an SIV virus or an HIV- 1 virus.

6. A composition according to any one of the preceding claims wherein the immunogen derivable from the first virus is an Env gp l40 protein and the immunogen derivable from the second virus is an Env gp l40 protein or a polynucleotide capable of expressing a gp l40 protein.

7. A composition according to any one of the preceding claims wherein the immunogen derivable from the second virus is a gp l40 trimeric Env protein or a polynucleotide capable of expressing a gp l40 protein.

8. A composition according to any one of the preceding claims wherein the immunogen derivable from the second virus is a gp l40 trimeric Env protein from SIVmac239 or HIV-2 or a polynucleotide capable of expressing a gp l40 trimeric Env protein from SIVmac239 or HIV-2.

9. A composition according to any one of the preceding claims wherein the immunogen derivable from the second virus is:

a) a protein having the amino acid sequence set out in SEQ ID 2 or SEQ ID 3 ; b) a protein having the amino acid sequence of amino acids 30 to 676 of SEQ ID NO 2 or of amino acids 3 1 to 677 of SEQ ID NO. 3 ;

c) a protein having 90% sequence identity to a) or b); or

c) a polynucleotide capable of expressing a protein of a), b) or c) .

10. A composition according to any one of the preceding claims wherein the immunogen derivable from the first virus has an amino acid sequence as set out in SEQ ID NO. 1 or an amino acid sequence as set out in amino acids 30 to 655 of SEQ ID NO. 1.

1 1. A composition according to any one of the preceding claims formulated for simultaneous or sequential administration with a further composition comprising the immunogen derivable from the first virus.

12. A composition according to claim 1 1 wherein the immunogenic composition comprising an immunogen derivable from the first virus is administered before, after or simultaneously with the composition comprising an immunogen derivable from the second virus.

13. A composition according to claim 1 1 or claim 12 wherein the immunogen derivable from the first virus is an Env protein or polynuclotide capable of expressing an Env protein.

14. A composition according to any one of claims 1 1 to 13 wherein the first virus is HIV- 1 and the immunogen derivable from the first virus is a mixture of 2- 7 different env genes; and the second virus is SIVmac239 and the immunogen derivable from the second virus is a SIVmac239 gp l40 trimer.

15. A composition according to any one of claims 1 1 to 14 wherein the first virus is HIV- 1 and the immunogen derivable from the first virus is a mixture of 3 different env genes; and the second virus is SIVmac239 and the immunogen derivable from the second virus is a SIVmac239 gp l40 trimer.

16. A composition according to any one of claims 1 1 to 15 wherein the first virus is HIV- 1 and the immunogen derivable from the first virus is a mixture of three different env genes from clade B; and the second virus is SIVmac239 and the immunogen derivable from the second virus is a SIVmac239 gp l40 trimer.

17. A composition according to any one of claims 1 to 16 for use with an anti- HIV- 1 T-cell immunogen.

18. A method of inducing an immune response in an organism to a first virus comprising the step of administering to the organism an immunogenic composition comprising an immunogen derivable from a second virus, wherein the second virus has from 30% and 90% protein sequence identity to an equivalent immunogen derivable from the first virus.

19. A method according to claim 18 wherein the second virus has between 40% and 80% protein sequence identity similarity to the first virus.

20. A method according to claim 18 wherein the immunogen derivable from the second virus has between 40% and 80 % identity to the immunogen derivable from the first virus.

21. A method according to any one of claims 18 to 20 wherein the immune response is a neutralising antibody response.

22. A method according to any one of claims 18 to 21 wherein the first virus is an HIV- 1 virus and the second virus is an SIV virus or an HIV-2 virus.

23. A method according to any one of claims 18 to 22 wherein the first virus is an HIV-2 virus and the second virus is an SIV virus or an HIV- 1 virus.

24. A method according to any one of claims 18 to 23 wherein the immunogen derivable from the second virus is a gp l40 protein or a polynucleotide capable of expressing a gp l40 protein.

25. A method according to any one of claims 18 to 24 wherein the immunogen derivable from the second virus is a gp l40 trimeric Env protein or a polynucleotide capable of expressing a gp l40 protein.

26. A method according to any one of claims 18 to 25 wherein the immunogen derivable from the second virus is a gp l40 trimeric Env protein from SIVmac239 or HIV-2 or a polynucleotide capable of expressing a gp l40 trimeric Env protein from SIVmac239 or HIV-2.

27. A method according to any one of claims 18 to 26 wherein the immunogen derivable from the second virus is a) a protein having the amino acid sequence set out in SEQ ID 4 or SEQ ID6; b) a protein having 90% sequence identity to a); or a polynucleotide capable of expressing a protein of a) or b).

28. A method according to any one of claims 18 to 27 further comprising a step of administering to the organism a second immunogenic composition comprising an immunogen derived or derivable from the first virus.

29. A method according to claim 28 wherein the immunogenic composition comprising an immunogen derivable from the first virus is administered before, after or simultaneously with the composition comprising an immunogen derivable from the second virus.

30. A method according to claim 28 or claim 29 wherein the immunogen derivable from the first virus is an Env protein or polynuclotide capable of expressing an Env protein.

3 1. A method according to any one of claims 28 to 30 wherein the first virus is HIV- 1 and the first immunogen is a mixture of three different env genes from clade B; and the second virus is SIVmac239 and the second immunogen is a SIVmac239 gp l40 trimer.

32. An anti-HIV-1 or anti-HIV-2 antibody produced by the method of any one of claims 18 to 31.

33. An immunogenic composition comprising an immunogen derivable from an HIV-1 virus and a) an immunogen derivable from an SIV virus; and/or an immunogen derivable from an HIV-2 virus for raising an immune response against HIV-1 virus.

34. A kit comprising (i) a priming composition comprising an immunogen derivable from an HIV-1 virus; (ii) a boosting composition comprising either or both of; an immunogen derivable from an SIV virus and an immunogen derivable from an HIV-2 virus; (iii) instructions to administer the priming composition and the boosting composition to an organism.

35. Use of a composition comprising an immunogen derivable from an SIV virus and/or a composition comprising an immunogen derivable from an HIV-2 virus for raising an immune response against HIV-1.

36. A composition, method, use or kit according to any preceding claim wherein the immunogens are administered with an adjuvant.

37. A composition, method, use or kit according to any preceding claim wherein the immunogens are co-administered with another immunogenic composition or vaccine.