Patent Application
20160122395
The present specification teaches in the general field of pathogenic viruses. More particularly, the specification relates to human immunodeficiency virus (HIV) vaccines and related fields. In one particular aspect, the specification relates to modified HIV envelope glycoproteins (Env) and provides a process for modifying HIV Env-based immunogens for use in vaccine protocols to enhance the ability of a subject to produce broadly neutralizing antibodies (brNAbs).
This application contains a Sequence Listing which is submitted herewith in electronically readable format. The electronic Sequence Listing file was created on Jan. 6, 2016, is named “376507_ST25.txt” and had a size of 35.9 KB. The entire contents of the Sequence Listing in the electronic “376507_ST25.txt” file are incorporated herein by this reference.
Bibliographic details of references referred to by number in Example 1 and Example 2 are listed at the end of the Examples under “Bibliography 1” and “Bibliography 2”, respectively. Bibliography details of references referred to by author are listed at the end of the Examples as “Bibliography 3”.
Bibliography details of references referred to in Table A are listed under Table A.
The reference to any prior art is not and should not be taken as an acknowledgment or any form of suggestion that this prior art forms part of the common general knowledge.
There has long been an interest in the ability of certain host immunoglobulins (antibodies) to reduce or block (neutralize) the ability of viral pathogens to initiate or perpetuate an infection in the host. For example, in the case of hepatitis B virus, administration of anti-hepatitis-B virus antibodies within twelve hours of birth to infants born to infected mothers is effective in preventing mother to infant transmission of the virus. Coupled with an active HBV vaccine this strategy is 98% effective (Beasley et al., Lancet, 2: 1099-102, 1983). For HIV Rubrecht et al. (Rubrecht et al., Vaccine. 21: 3370-3, 2003) showed that passive transfer of neutralizing monoclonal antibodies that were available in 2003 (i.e., neutralizing monoclonal antibodies b12, 2G12, 2F5 and 4E10) was effective in protecting macaques against a clade B primary HIV isolate.
While it is possible for passive transfer of neutralizing antibodies to be an effective treatment or preventative approach, it is currently considered that the induction of a broad neutralizing antibody response will be a critical ability of any effective vaccine. A major perceived obstacle to a vaccine against HIV has been its high mutation rate leading to multiple different genetic forms of virus and more particularly enhancing the diversity within the Env glycoprotein which is the target of many neutralizing antibodies. Based upon genetic similarities, HIV-1 viruses (which cause infections in man more commonly than HIV-2) are grouped into four groups, M, O, N and P. M is the major group and this group has been classified into different geographically and genetically distinct clades, clades A through to H, J, and K.
There are examples of chronically infected patients who have over time developed serum antibodies to Env that neutralize virus from diverse HIV clades. Most of the early neutralizing antibodies identified display activity predominantly against neutralization sensitive HIV strains (tier 1 strains) while most circulating HIV strains are less sensitive to neutralizing antibodies (tier 2 strains). Thus, an effective vaccine is thought to be one which induces broadly neutralizing antibodies against multiple clades of HIV, or at least against clade C HIV which predominates world wide and has caused more than half of HIV infections. Furthermore, vaccines which engender antibodies that recognise tier 1 and tier 2 strains are also sought.
An effective HIV vaccine remains an elusive goal. HIV infections continue to cause millions of deaths around the world every year and since the HIV was recognised in 1990 as the causative agent of the AIDS epidemic, over 30 million people have died from AIDS-related causes. The World Health Organisation conservatively estimates that there were 34 million people living with HIV/AIDS in 2010 with 2.7 million people newly infected in that year of whom 14% were children. The mainstay of successful treatment is combination anti-retroviral drug therapy (cART) which can slow disease progression through viral suppression. However, cART but has serious side effects.
More recently, new, highly potent, and broadly neutralizing antibodies have been identified. The most powerful method for identifying neutralizing antibodies has been to sort individual memory B cells from rare individuals who display broadly neutralising antibodies (brNAbs) followed by micro neutralization assays. These antibodies are directed against the CD4 binding site, to the membrane proximal ectodomain region (MPER) of gp41 and to oligomannose glycan-dependent epitopes. The following table illustrates a range of neutralizing antibodies, their target sites and their neutralizing capacity.
1Walker et al., Nature 477: 466-70, 2011
2Walker et al., Science 326: 285-9, 2009.
3Huang et al., Nature 491: 406-12, 2012.
As noted above, passive transfer of various brNAbs individually or in combination has been shown to confer protection in macaque. Illustrative examples include the use of b12 (Burton et al., Proc Natl Acad Sci USA 108: 11181-6, 2011; Veazy et al., Nat Med 9: 343-6, 2003) 2G12 (Hessell et al., PLOS pathog 5: e1000433, 2009), PGT121 (Moldt et al., Proc Natl Acad Sci USA 109: 18921-5, 2012) and b12/2G12/2F5/4E10 combinations (Ruprecht et al., Vaccine. 21: 3370-3, 2003). It is also relevant to note that Ibalizumab (αCD4bs) monotherapy of HIV-1 infected individuals leads to a transient drop in viral load to nadir levels followed by a rebound in viral titre due to emergence of resistant mutants (Bruno & Jacobson, Antimicrob Chemother 65: 1839-41, 2010; Toma et al J Virol 85: 3872-80, 2011).
Studies show that these potent neutralizing antibodies, determined by Env glycoprotein epitopes, are critical in providing protection against viral challenge and it is hoped that further study of the epitopes in gp120/gp41 may provide better Env-based immunogens. However, it is unknown how to present these epitope in a format that will be any more effective in generating neutralizing antibodies than current vaccine Env-based immunogens. For example, three of the most broadly reactive neutralizing antibodies against HIV (2F5, 4E10 and Z13) bind to the membrane proximal ectodomain region (MPER) and contribute to protection yet the design of a vaccine which elicits antibodies with the same specificities has proven difficult.
Given the lack of effective therapies for the treatment or prevention of HIV infection there is an urgent need for immunogens and vectors capable of engendering immune responses effective in preventing or reducing HIV infection.
The present disclosure is predicated in part on the experimental and theoretical determination that forced evolution of an attenuated virus, having a mutation in the viral complex that mediates viral fusion and host cell entry, provides a process for producing second site suppressor mutants likely to serve as enhanced immunogens for the production of neutralizing antibodies targeting the complex. The effectiveness of this strategy is illustrated in Example 1 which describes a second site mutation in the conserved membrane proximal ectodomain region (MPER) of HIV-1 glycoprotein (gp) 41. The MPER mutant displays increased sensitivity of gp41 MPER dependent neutralizing antibodies. Example 2 provides a second site mutation in the external glycan shield region (comprising asparagine-linked oligosaccharides) of glycoprotein (gp) 120 variable region 1 (V1). The V1 glycosylation mutant displays increased sensitivity of gp120 glycan dependent neutralizing antibodies. Example 3 shows how these two mutants have been combined to produce an Env mutant displaying enhanced sensitivities against each of the broadly neutralizing antibodies 2G12, PGT121, PGT126 and 4E10 (see Table A and
While described with respect to HIV, the present invention extends to any lentivirus envelope immunogen such as one in respect to FIV, SIV and BIV. Furthermore, the invention extends to forced evolution of virus envelope protein selected from the group comprising a Flavivirus (e.g. hepatitis C virus), Coronavirus, Herpesvirus, Hepadnavirus, Retrovirus (including HIV), Orthomyxovirus (e.g., influenza) or Paramyxovirus (e.g. measles virus) envelope proteins.
The HIV-1 envelope glycoprotein complex comprises a trimer of gp120 subunits in non-covalent association with a trimer of transmembrane gp41 subunits and mediates viral attachment membrane fusion and viral entry. A gp120-gp41 association site is formed by the terminal segments of C1 and C5 of gp120 and the central disulfide-bonded region of gp41 (see
Reference to a “second site suppressor” mutation refers herein to Env comprising at least “a second mutation” wherein the second mutation affects the phenotype which is caused by a first pre-determined mutation at a distinct location to the second mutation. In accordance with the present invention, the first mutation is a mutation in the gp120-gp41 association site which attenuates the viral particle such that cell to cell transmission and cell free virus infectivity is disabled.
In some embodiments, the association site mutation forms a pseudoreversion or reversion mutant and this embodiment is also encompassed. In some embodiments, the first mutation is deleterious and the second mutation or the pseudoreversion mutation complements the phenotype of the first mutation. While the invention is described with respect to second site mutations, the skilled person will understand that the invention is not limited to a “second” site and that “third” or “fourth” etc site mutations may be employed.
In some embodiments, the process comprises assessing the sensitivity of the transmission competent variant (TCV) to a neutralizing antibody. As known to those of skill in the art neutralization assays may be achieved using a number of different protocols. While the entire TCV from (ii) may be tested, it will be apparent that any variant comprising the gp120-gp41 association site mutation or a reversion mutation and/or a second site suppressor mutation may be assayed. In some embodiments, the second site mutation may be transferred into an infectious or pseudotyped virus particle for assessment.
In some embodiments, the gp120-gp41 association site mutant comprises a mutation in the central disulfide bonded region (DSR) of gp41 as alignment of the DSR from a range of HIV types is set out in
In some embodiments, the modified Env immunogen binds preferentially to neutralizing antibodies that recognise the V1/V2 region of gp120. In other embodiments, the modified Env immunogen binds preferentially to neutralizing antibodies that bind to the V3 region of gp120. In other embodiments, the modified Env immunogen binds preferentially to neutralizing antibodies that bind to the MPER region of gp41. In some embodiments, the modified Env immunogen binds preferentially to neutralizing antibodies that recognise the V1/V2 region of gp120 and the MPER region of gp41.
In one embodiment, the specification enables a modified HIV envelope glycoprotein (Env) antigen or a lipid containing vehicle comprising same wherein the Env antigen comprises one of: (i) a second site suppressor mutation in residue 674 of the membrane proximal ectodomain region (MPER) of HIV gp41; (ii) a second site suppressor mutation which ablates a glycosylation site in the variable region (V1) region of gp120; or (iii) a second site suppressor mutation ablating a glycosylation site in the V1 region of gp120 and a second site suppressor mutation in residue 674 of the MPER of HIV gp41.
In another aspect, the present invention provides a modified HIV Env immunogen wherein the Env immunogen comprises: (i) a gp120-gp41 association site mutation or a reversion or pseudoreversion mutation thereof and a second site suppressor mutation in MPER; (ii) a gp120-gp41 association site mutation or a reversion or pseudoreversion mutation thereof and a second site suppressor mutation in a glycosylation site in the V1 region; or (iii) a gp120-gp41 association site mutation or a reversion or pseudoreversion mutation thereof and a second site suppressor mutation in a glycosylation site in the V1 region and a second site suppressor mutation in MPER.
Reference herein “MPER” means the conserved 23-residue tryptophan-rich domain which connects the helical region 2 (HR2) of the gp41 ectodomain to the transmembrane domain.
In certain embodiments the Env antigen comprises a gp120-gp41 association site mutation or a reversion or pseudoreversion mutation thereof in the disulfide bonded region (DSR) of gp120, preferrably at K601 and/or W596.
Examples of the mutation at K601 and W596 include K601D, K601H, K601N, K601Q and K601R, and W596I, W596L, W596H, W596M, W596Y, W596F and W596A.
It is also preferred that the Env antigen comprises a mutation such that residue 674 is other than aspartic acid and is preferrably is glutamic acid.
Glycosylation site mutations in V1 of HIV gp120 are preferably ΔN139INN or T138N or a mutation of asparagine(s), threonine(s) or serine(s) in other HIV strains that ablate analogous glycosylation sites.
The lipid vehicle may be a human immunodeficiency virus like particle (HIVLP) or an enveloped virus or virus-like particle that is other than human immunodeficiency virus. Examples of relevant non HIV viruses include SIV, murine leukemia virus and other retroviruses, vesicular stomatitis virus, rabies virus, herpesvirus and hepadnavirus. Clearly the lipid vehicle may also be a non-viral lipid.
In another aspect the present invention provides a modified Env antigen comprises a mutation selected from the group consisting of ΔN139INN/W596L/K601H/D674E, ΔN139INN/W596L/K601D/D674E, ΔN139INN/W596L/K601N/D674E, W596L/K601H/D674E, ΔN139INN/W596L/K601H, T138N/W596L/K601H/D674E, T138N/ΔN139INN, T138N, ΔN139INN and a mutation of asparagine(s), threonine(s) or serine(s) in other HIV strains that ablate analogous glycosylation sites.
In yet another aspect the present invention provides an isolated nucleic acid molecule encoding the modified Env antigen of the present invention.
In a still further aspect the present invention provides a composition comprising the Env antigen or lipid vehicle of the present invention a pharmaceutically or physiologically acceptable carrier or diluent. The composition may also comprise other HIV antigens.
The present invention also provides a method of eliciting an immune response in a subject, the method comprising administering an effective amount of a composition according to the present invention for a time and under conditions sufficient to elicit an immune response. The immune response may comprise the production of neutralizing antibodies or the production of antibodies that prevent HIV replication through mechanisms other than neutralization.
In some embodiments, the modified or isolated Env immunogen is provided in a lipid containing vehicle such as a virus-like particle (VLP) or other lipid containing vehicle.
In some embodiments, the gp120-gp41 association site mutation is a DSR mutation. In an illustrative embodiment, the DSR mutation is in K601 such as K601D or a conservative substitution thereof (e.g. K601E). Exemplary substitutions are set out in Table 2. In some embodiments, the association site mutation reversion or pseudoreversion is K601K (reversion), K601H (pseudoreversion) or K601N (pseudoreversion) or a conservative substitution for lysine such as glutamine (K601Q) or arginine (K601R).
In some embodiments, the MPER mutation is D674E of HIV gp41. In one illustrative embodiment the Env immunogen comprises the MPER mutation D674E together with the DSR pseudoreversion K601H/N and the DSR mutation W596L.
In some embodiments, mutation in VI of HIV gp120 is a glycosylation site mutation.
In some embodiments, mutation in V1 of HIV gp120 is the glycosylation mutant, ΔN139INN. This deletion ablates overlapping Asn141-Asn142-Ser-Ser potential N-linked glycosylation sequons (PNGS) in V1. Corresponding mutations are made to conserved asparagine-rich portions of V1 of different strains. An alignment of the V1 regions from multiple strains is set out in
In other embodiments the mutation in V1 of HIV gp120 is the glycosylation mutant, T138N. This substitution ablates the Asn136 potential N-linked glycosylation sequons (PNGS) in V1.
In some embodiments, the present invention provides a nucleic acid molecule encoding a modified HIV Env immunogen of the present invention, plasmids, expression vectors and cells comprising same. In one illustrative embodiment, the nucleic acid molecule encodes an Env immunogen comprising W596L and K601H substitutions in the DSR of gp41, a substitution D674E in the MPER of gp41 and a deletion comprising ΔN139INN of V1 of gp120. Corresponding glycosylation site mutations include mutation/ablation of one or two PNGS in V1, as highlighted in
The invention provides an Env immunogen or a nucleic acid molecule encoding same identified, produced or selected by the process described herein for identifying, producing or selecting modified Env immunogens.
In some embodiments, the present invention provides a composition comprising a remodelled Env immunogen or a vector encoding same as described herein and a pharmaceutically or physiologically acceptable carrier or diluent.
In some embodiments, the composition is for use in therapy, such as HIV prophylaxis or treatment.
Vaccine compositions are contemplated comprising a HIV Env immunogen in a lipid containing particle as an immunologically active component. Vaccine compositions (vaccines) may also contain additional components to enhance the immunological activity of the active component in a mammalian subject, such as an adjuvant.
In another embodiments, the present invention provides the composition in, or in the manufacture of a medicament for, the treatment or prevention of HIV infection.
Also contemplated are kits or solid substrates comprising a remodelled Env immunogen as described herein or a lipid containing complex comprising same.
The above summary is not and should not be seen in any way as an exhaustive recitation of all embodiments of the present invention.
If figures contain colour representations or entities, coloured versions of the figures are available from the Patentee upon request or from an appropriate Patent Office. A fee may be imposed if obtained from a Patent Office.
Table A illustrates available neutralizing antibodies, their specificity in Env and their neutralizing potency and breadth.
Table 1 provides an amino acid sub-classification.
Table 2 provides exemplary conservative amino acid substitutions.
The subject invention is not limited to particular screening procedures for agents, specific formulations of agents and various medical methodologies, as such may vary.
Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs. Any materials and methods similar or equivalent to those described herein can be used to practice or test the present invention. Practitioners are particularly directed to Sambrook et al., 1989 Chapters 16 and 17, Coligan et al., Current Protocols In Protein Science, John Wiley & Sons, Inc., 1995-1997, in particular Chapters 1, 5 and 6. and Ausubel et al., Current Protocols in Molecular Biology, Supplement 47, John Wiley & Sons, New York, 1999; Colowick and Kaplan, eds., Methods In Enzymology, Academic Press, Inc.; Weir and Blackwell, eds., Handbook of Experimental Immunology, Vols. I-IV, Blackwell Scientific Publications, 1986; Joklik ed., Virology, 3rd Edition, 1988; Fields and Knipe, eds, Fundamental Virology, 2nd Edition, 1991; Fields et al., eds, Virology, 3rd Edition, Lippincott-Raven, Philadelphia, Pa., 1996, for definitions and terms of the art and other methods known to the person skilled in the art.
Throughout this specification and the claims which follow, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.
As used herein the singular forms “a”, “an” and “the” include plural aspects unless the context clearly dictates otherwise.
Each embodiment in this specification is to be applied mutatis mutandis to every other embodiment unless expressly stated otherwise.
Thus, in one aspect of the present invention, a process is provided for producing a modified or isolated immunogen to confer a neutralizing antibody response to a target antigen in a mammalian subject which is enhanced over that conferred by the unmodified immunogen. In one embodiment relating to HIV Env, the process generally comprises the following steps: (i) maintaining a cell to cell transmission attenuated HIV particle comprising a gp120-gp41 association site mutation which attenuates cell to cell transmission in a permissive cell for a time sufficient to promote the development of a cell to cell transmission competent variant (TCV). In some embodiments, the process comprises (ii) isolating a cell to cell transmission replication competent variant (TCV) HIV particle from (i). In another embodiment, the process comprises (iii) determining the amino acid sequence of all or part of Env from the TCV in step (ii) to identify a second site suppressor mutation.
Reference to a “second site suppressor” includes a second mutation distant from a first mutation wherein the second mutation affects the phenotype which is caused by the first mutation. In some embodiments, the first mutation is deleterious and the second mutation complements the phenotype of the first mutation.
Reference to “enhanced” includes qualitative as well as quantitative improvement relative to a control (unmodified) immunogen. This may be determined using any method in the art such as by determining the epitope specificity, cross-clade sensitivity, IC50 or IC90s or percentage neutralisation by monoclonal or polyclonal antibodies (such as sera from an immunised subject). In some embodiments, neutralisation potency, breadth or sensitivity is improved by at least 20%, 40%, 60%, 90%, 100%, 200%, 300%, 400% etc. Qualitative differences can be determined readily such as by comparing the absence of neutralizing antibody to a particular epitope, moiety or region of Env compared to the presence of neutralizing antibody to that epitope, moiety or domain.
Reference to maintaining includes passage and serial passage of replication defective virus in a permissive cell. Suitable cells or cell lines are known in the art and include PMBC and U87 cells comprising CD4 and an appropriate viral co-receptor.
In some embodiments, the process comprises assessing the sensitivity of the transmission competent variant (TCV) to a neutralizing antibody. As known to those of skill in the art neutralization assays may be achieved using a number of different protocols. While the entire TCV from (ii) may be tested, it will be apparent that any TCV comprising the gp120-gp41 association site mutation and the second site suppressor mutation may be assayed. In some embodiments, the second site mutation may be transferred into another infectious particle for testing. In this way the mutations may be assessed against a background of at least two and preferably multiple clades and tiers of virus.
Neutralization assays are generally conducted using a panel of neutralizing agents such as antibodies as described in the examples. Exemplary monoclonal antibodies are PG16, 2G12, b12, 2F5, 4E10, VRC01, PGT121/8, PGT145 and 10E8.
Neutralization sensitivity assays are generally conducted to determine the ability of the modified Env in the context of a virion or lipid-containing vehicle to be neutralized by any at least two neutralizing antibodies recognising a combination of specificities (e.g., CD4bs and gp41MPER), (ii) by neutralizing antibodies that neutralise tier 1 and tier 2 isolates (ii) by neutralising antibodies that neutralise one or more HIV clades of interest; and to the potency of the neutralization with neutralizing antibodies (i.e. comparing the IC50 or IC90 values with control HIV particles selected from neutralisation sensitive or neutralization resistance particles known in the art.
Immunisation protocols are known to the skilled address and described herein. These may include the use of a range of adjuvants, sustained release formulations and administration protocols designed to test the ability of the remodelled Env immunogen to elicit an immune response, and preferably a neutralizing antibody response. Cross-clade neutralization assays are also known in the art and are contemplated herein.
In some embodiments, gp120-gp41 association site mutant comprises a mutation in the disulfide bonded region (DSR) of gp41. Alternative association site mutants may comprise mutations in the N-terminal or C-terminal segments of gp120. In one embodiment, the DSR mutation is of K601 within the DSR. In some embodiments, the K601 mutation is K601D.
In some embodiments, the process is conducted in vitro.
In some embodiments the virus is HIV, preferably HIV-1.
In some embodiments, the neutralizing antibody is a gp120 glycan directed antibody. The epitope recognised by a particular antibody is determined using standard art recognised protocols.
In other embodiments, the neutralizing antibody is a gp41 MPER directed antibody.
In further embodiments, the neutralizing antibody is a CD4 binding site directed antibody.
In a further embodiment, the process comprises assessing the sensitivity of the TCV or a virus comprising second site suppressor mutation to human sera or antibodies selected therefrom, or another potential anti-HIV agent, such as a fusion inhibitor peptide.
In some embodiments, the part of Env is the V1 domain of HIV gp120.
In some embodiments, the part of Env is the MPER domain of HIV gp41.
In some embodiments, a modified HIV envelope glycoprotein (Env) antigen or a lipid containing vehicle comprising same wherein the Env antigen comprises one of: (i) a second site suppressor mutation in residue 674 of the membrane proximal ectodomain region (MPER) of HIV gp41; (ii) a second site suppressor mutation which ablates a glycosylation site in the variable region (V1) region of gp120; or (iii) a second site suppressor mutation ablating a glycosylation site in the V1 region of gp120 and a second site suppressor mutation in residue 674 of the MPER of HIV gp41.
In another aspect, the present invention provides a modified HIV Env immunogen wherein the Env immunogen comprises: (i) a gp120-gp41 association site mutation or a reversion or pseudoreversion mutation thereof and/or a second site suppressor mutation in MPER; (ii) a gp120-gp41 association site mutation or a reversion or pseudoreversion mutation thereof and/or a second site suppressor mutation in a glycosylation site in the V1 region; or (iii) a gp120-gp41 association site or a reversion or pseudoreversion mutation thereof mutation and/or a second site suppressor mutation in a glycosylation site in the V1 region and a second site suppressor mutation in MPER.
Reference to Env immunogen or Env or Env polypeptide and the like includes an Env glycoprotein from any genotype, group, clade or isolate of HIV. The term further includes non-naturally occurring variants including portions of the full length Env provided they are able to display the portions of the glycoprotein necessary to induce a neutralizing immune response in a subject.
In nature, the Env complex comprises a trimer of gp120 subunits in non-covalent association with a trimer of transmembrane gp41 subunits and this complex mediates viral attachment, membrane fusion and viral entry (for review see 1 in Bibliography 2). Within gp120, five conserved regions (C1-C5) alternate with five variable regions (V1-V5). The conserved regions largely form the gp120 core comprising inner and outer subdomains that are bridged by four antiparallel β-strands (the bridging sheet), whereas the variable regions form external solvent-exposed loops (see 3, 4, 5, 6, 7, 8 in Bibliography 2). gp120 is anchored to the viral envelope by the trimeric transmembrane/fusion glycoprotein, gp41. The ectodomain of gp41 comprises an N-terminal fusion peptide linked through N- and C-terminal α-helical heptad repeat sequences (HR1 and HR2, respectively) to a C-terminal membrane anchor and cytoplasmic tail. A central disulfide-bonded loop region or DSR joins HR1 to HR2 (See
The membrane fusion and viral entry function of gp120-gp41 involves conformational changes that are triggered by receptors. CD4 ligation is believed to reorganize V1V2 and V3 to expose a binding site for the chemokine receptors CCR5 and CXCR4, which function as fusion cofactors (see 3, 4, 5, 6, 9, 10, 11, 12 in Bibliography 2). The V3 loop mediates important contacts with the negatively charged N-terminal domain and extracellular loop 2 of CCR5 and CXCR4 and determines the chemokine receptor preference of HIV-1 isolates. In a virion context, CD4 binding causes an “opening up” of the gp120 trimer due to outward rotation and displacement of gp120 monomers (see 10, 12 in Bibliography 2).
The gp120-receptor interactions cause gp41 to transition from a dormant metastable structure into a fusion active state (see 1, 2, 13, 14 in Bibliography 2). Structural transitions in gp41 that are associated with fusion function include the insertion of the fusion peptide into the target membrane and formation of a “prehairpin intermediate” structure wherein a triple-stranded coiled coil of HR1 segments provides a binding surface for the HR2 (see 15, 16, 17, 18, 10 in Bibliography 2). Antiparallel HR1-HR2 interactions forma 6-helix bundle which opposes the N- and C-terminal membrane inserted ends of gp41, and the associated viral and cellular membranes, leading to merger and pore formation (see 20, 21, 22, 23, 24 in Bibliography 2).
How conformational signals are transmitted between receptor-bound gp120 and gp41 to trigger the refolding of gp41 into the fusion-active state is being elucidated. A gp120-gp41 association site formed by the terminal segments of C1 and C5 of gp120 and the central DSR of gp41 (see 25, 26, 27, 28 in Bibliography 2) may play an important role in this process as mutations in the DSR can inhibit CD4-triggered gp41 prehairpin formation and the initial hemifusion event (see 29 in Bibliography 2). Furthermore, the introduction of Cys residues to C5 and to the DSR generates an inactive disulfide-linked gp120-gp41 complex that is converted to a fusion-competent form by reduction (see 30, 31 in Bibliography 2). These findings implicate the C1-C5-DSR synapse in maintaining gp120-gp41 in the prefusion state and in subsequent transmission of fusion activation signals emanating from receptor-bound gp120. The terminal C1 and C5 gp41-contact regions project ˜35-Å from a 7-stranded β-sandwich at the base of the gp120 inner domain (8 in Bibliography 2). This β-sandwich appears to also play an important role in conformational signalling between gp120 and gp41 by linking CD4-induced structural changes in three structural layers of gp120 that emanate from the β-sandwich to gp41 activation (32 in Bibliography 2).
Understanding how conserved functional determinants of the HIV-1 glycoproteins tolerate or adapt to the rapid evolution of other Env regions is important for their evaluation and exploitation as potential drug and/or vaccine targets. For example, mutations in the DSR confer resistance to a novel low molecular weight fusion inhibitor, PF-68742, implicating this gp41 region as an inhibitor target (33 in Bibliography 2). Neutralizing antibodies exert strong evolutionary pressures on Env that can result in an increase in the number and/or a change in the position of potential N-linked glycosylation sites (PNGSs) that modify NAb-Env interactions (34, 35 in Bibliography 2). V1V2 is a key regulator of neutralization resistance, which generally correlates with its elongation and acquisition of PNGSs (34, 36, 37, 38, 39, 40, 41, 42, 43, 44 in Bibliography 2). Previously, the C1-C5-DSR association site was identified as a conserved determinant that exhibits structural and functional plasticity. This idea is based on the finding that whereas the overall gp120-gp41 association function of the DSR is conserved, the contribution of individual DSR residues to gp120 anchoring and membrane fusion function varies among HIV-1 strains and is controlled by V1V2 and V3 (28 in Bibliography 2. It is proposed that this plasticity enables the maintenance of a functional glycoprotein complex in a setting of host selective pressures that drive the rapid coevolution of gp120 and gp41.
Reference herein “MPER” means the conserved 23-residue Trp-rich sequence that connects the helical region 2 (HR2) of the gp41 ectodomain to the transmembrance domain.
Env immunogens may be produced by recombinant means typically in eukaryotic cells using methods known in the art. Eukaryotic cells include mammalian, plant, yeast and insect cells as known in the art. Recombinant proteins are produced by culturing the host cells for a period of time sufficient to allow for expression of the protein in the host cells or, more preferably, secretion of the protein into the culture medium in which the host cells are grown.
“Recombinant host cells”, “host cells”, “cells”, “cell lines”, “cell cultures”, and other such terms denoting prokaryotic microorganisms or eukaryotic cell lines cultured as unicellular entities, are used interchangeably, and refer to cells which can be, or have been, used as recipients for recombinant vectors or other transfer DNA, and include the progeny of the original cell which has been transfected.
Suitable mammalian cell lines include, but are not limited to, BHK, VERO, HT1080, 293, 293T, 293F, RD, COS-7, CHO, Jurkat, HUT, SUPT, C8166, MOLT4/clone8, MT-2, MT-4, H9, PM1, CEM, myeloma cells (e.g., SB20 cells) and CEMX174 are available, for example, from the ATCC. Other host cells include without limitation yeast, e.g. Pichia pastoris, or insect cells such as Sf9 cells.
Synthetic DNA may be recombinantly expressed by molecular cloning into an expression vector containing a suitable promoter and other appropriate transcription regulatory elements, and transferred into prokaryotic or eukaryotic host cells to produce recombinant protein. Techniques for such manipulations are described by Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd ed.). Cold Spring Harbour Laboratory, Cold Spring Harbour, N Y, 1989; Ausubel et al., Current Protocols in Molecular Biology, Green Pub. Associates and Wiley-Interscience, New York, 1988.
For example, a construct for expression in yeast preferably contains a synthetic gene, with related transcriptional and translational control sequences operatively linked to it, such as a promoter (such as GAL10, GALT, ADH1, TDH3 or PGK), and termination sequences (such as the S. cerevisiae ADH1 terminator). The yeast may be selected from the group consisting of: Saccharomyces cerevisiae, Hansenula polymorpha, Pichia pastoris, Kluyveromyces Kluyveromyces lactis, and Schizosaccharomyces pombe. See also Yeast Genetics: Rose et al., A Laboratory Course Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y., 1990. Nucleic acid molecules can be codon optimized for expression in yeast as known in the art (see Sharp and Cowe, Yeast, 7: 657-678, 1991). Appropriate vectors and control elements for any given cell type can be selected by one having ordinary skill in the art in view of the teachings of the present specification and information known in the art about expression vectors.
Vectors available for cloning and expression in host cell lines are well known in the art, and include but are not limited to vectors for cloning and expression in mammalian cell lines or yeast (fungal) cells, vectors for cloning and expression in bacterial cell lines, vectors for cloning and expression in phage and vectors for cloning and expression in insect cell lines. The expressed proteins can be recovered using standard protein purification methods.
Translational control elements have been reviewed by M. Kozak (e.g., Kozak, Mamm Genome, 7(8): 563-74, 1996; Kozak, Biochimie., 76(9): 815-21, 1994; Kozak, J Cell Biol, 108(2): 229-241, 1989; Kozak and Shatkin, Methods Enzymol, 60: 360-375, 1979).
Recombinant glycoproteins can be conveniently prepared using standard protocols as described for example in Sambrook, et al., 1989 (supra), in particular Sections 13, 16 and 17; Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons Inc, 1994, in particular Chapters 10 and 16; and Coligan et al., 1995-1997 (supra), in particular Chapters 1, 5 and 6. The polypeptides or polynucleotides may be synthesized by chemical synthesis, e.g., using solution synthesis or solid phase synthesis as described, for example, in Chapter 9 of Atherton and Shephard, Peptide Synthesis. In Nicholson ed., Synthetic Vaccines, published by Blackwell Scientific Publications, and in Roberge et al., Science, 269(5221): 202-204, 1995.
In some embodiments, the modified Env immunogen is provided in a lipid containing vehicle such as a virus-like particle (VLP) or other lipid containing vehicle.
In one embodiment the specification provides a modified HIV envelope glycoprotein (Env) antigen or a lipid containing vehicle comprising same wherein the Env antigen comprises one of: (i) a second site suppressor mutation in residue 674 of the membrane proximal ectodomain region (MPER) of HIV gp41; (ii) a second site suppressor mutation which ablates a glycosylation site in the variable region (V1) region of gp120; or (iii) a second site suppressor mutation ablating a glycosylation site in the V1 region of gp120 and a second site suppressor mutation in residue 674 of the MPER of HIV gp41.
In another embodiment, the present invention provides a modified HIV Env immunogen or a lipid containing vehicle comprising same wherein the Env immunogen comprises one of: (i) a gp120-gp41 association site mutation or a reversion or pseudoreversion mutation thereof and/or a second site suppressor mutation in MPER; (ii) a gp120-gp41 association site mutation or a reversion or pseudoreversion mutation thereof and/or a second site suppressor mutation in a glycosylation site in the V1 region; or (iii) a gp120-gp41 association site mutation or a reversion or pseudoreversion mutation thereof and/or a second site suppressor mutation in a glycosylation site in the V1 region and a second site suppressor mutation in MPER.
In some embodiments, the gp120-gp41 association site mutation in the modified Env antigen is a DSR mutation or a reversion thereof or a pseudoreversion. In some embodiments, the reversion or pseudoreversion mutation permits cell to cell transmission competence in an intact viral particle. In some embodiments, the DSR mutation, reversion or pseudoreversion is K601H/N/K, i.e. to lysine, histidine or asparagine. Lysine is the wild-type residue, and histidine or asparagine are pseudoreversions. In some embodiments the W596L mutation is retained and allows cell to cell transmission in the context of K601H and D674E or ΔN179 INN.
In some embodiments, the MPER mutation is in D674E of HIV gp41.
In some embodiments, the glycosylation mutation in V1 of HIV is gp120 the Env antigen or lipid vehicle of any one of claims 1 to 4 wherein the glycosylation site mutation in V1 of HIV gp120 is loss of one or two potential N-linked glycosylation sites in asparagine(s) 141 and/or 142 of AD8 or a corresponding mutation in other HIV strains.
In some embodiments, the glycosylation mutation in V1 of HIV is the Env antigen or lipid vehicle of any one of claims 1 to 4 wherein the glycosylation site mutation in V1 of HIV gp120 is ΔN139INN or a corresponding deletion of conserved asparagine(s) in other HIV strains. See
In some embodiments, mutation in V1 of HIV gp120 is ΔN139INN.
In some embodiments the mutation in V1 is a glycosylation site mutation.
In some embodiments, the present invention provides a composition comprising a remodelled Env immunogen as described herein and a pharmaceutically or physiologically acceptable carrier or diluent.
Pharmaceutical compositions are conveniently prepared according to conventional pharmaceutical compounding techniques. See, for example, Remington's Pharmaceutical Sciences, 18th Ed., Mack Publishing, Company, Easton, Pa., U.S.A., 1990. These compositions may comprise, in addition to one of the active substances, a pharmaceutically acceptable excipient, carrier, buffer, stabilizer or other materials well known in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The carrier may take a wide variety of forms depending on the form of preparation desired for administration, e.g. intravenous, oral or parenteral. In some embodiments, the composition comprises an adjuvant.
The response of a mammalian subject to immunogens can be enhanced if they are administered as a mixture with one or more adjuvants. Immune adjuvants typically function in one or more of the following ways: (1) immunomodulation (2) enhanced presentation (3) CTL production (4) targeting; and/or (5) depot generation. Illustrative adjuvants include: particulate or non-particulate adjuvants, complete Freund's adjuvant (CFA), aluminium salts, emulsions, ISCOMS, LPS derivatives such as MPL and derivatives thereof such as 3D, mycobacterial derived proteins such as muramyl di- or tri-peptides, particular saponins from Quillaja saponaria, such as QS21 and ISCOPREP 703, ISCOMATRIX™ adjuvant, and peptides, such as thymosin alpha 1. An extensive description of adjuvants can be found in Cox and Coulter, “Advances in Adjuvant Technology and Application”, in Animal Parasite Control Utilizing Biotechnology, Chapter 4, Ed. Young, W. K., CRC Press 1992, and in Cox and Coulter, Vaccine 15(3): 248-256, 1997.
In some embodiments, the adjuvant is ISCOMATRIX.
In some embodiments, the composition is for use in therapy, such as HIV prophylaxis or treatment in a mammalian subject.
A mammalian subject for the purpose of treating an HIV infection includes a mammal including humans, primates, laboratory animals, domestic and farm animals, zoo, sport and pet animals. Preferably, the mammal is a human subject.
The term treatment refers to therapeutic measures taken to prevent or slow, reduce HIV infection and its associated disorders or symptoms or the risk of developing advanced symptoms of HIV infection, or reliance on cART, or reducing the side effects of cART by reducing the frequency with which cART medication must be taken to maintain low viral loads. The term refers to any measurable or statistically significant amelioration in at least some subjects in one or more symptoms of HIV infection, or in the risk of developing same or transmitting infection.
The term prophylaxis or prevention and the like include administration of a composition as described herein to a subject not known to be infected with HIV for the purpose of preventing or attenuating an infection or reducing the risk of becoming infected or reducing the severity or onset of a condition or signs of a condition associated with HIV infection such as AIDS or an AIDs related condition or infection.
The administration of the vaccine composition is generally for prophylactic purposes. The prophylactic administration of the composition serves to prevent or attenuate any subsequent infection. A “pharmacologically acceptable” composition is one tolerated by a recipient patient. It is contemplated that an effective amount of the vaccine is administered. An “effective amount” is an amount sufficient to achieve a desired biological effect such as to induce enough humoral or cellular immunity. This may be dependent upon the type of vaccine, the age, sex, health, and weight of the recipient. Examples of desired biological effects include, but are not limited to, production of no symptoms, reduction in symptoms, reduction in virus titre, complete or partial protection against infection by HIV. The terms “effective amount” including “therapeutically effective amount” and “prophylactically effective amount” as used herein mean a sufficient amount of a composition of the present invention either in a single dose or as part of a series or slow release system which provides the desired therapeutic, preventative, or physiological effect in some subjects. Undesirable effects, e.g. side effects, may sometimes manifest along with the desired therapeutic effect; hence, a practitioner balances the potential benefits against the potential risks in determining an appropriate “effective amount”. The exact amount of composition required will vary from subject to subject, depending on the species, age and general condition of the subject, mode of administration and the like. Thus, it may not be possible to specify an exact ‘effective amount’. However, an appropriate ‘effective amount’ in any individual case may be determined by one of ordinary skill in the art using routine skills or experimentation. One of ordinary skill in the art would be able to determine the required amounts based on such factors as prior administration of the compositions or other agents, the subject's size, the severity of a subject's symptoms or the severity of symptoms in an infected population, viral load, and the particular composition or route of administration selected.
In some embodiments, a vaccine or composition of the present invention is physiologically significant if its presence results in a detectable change in the physiology of a recipient patient that enhances or indicates an enhancement in at least one primary or secondary humoral or cellular immune response against at least one strain of HIV. The vaccine composition is administered to protect against viral infection. The “protection” need not be absolute, i.e., the HIV infection need not be totally prevented or eradicated, if there is a statistically significant improvement compared with a control population or set of patients. Protection may be limited to reducing the severity or rapidity of onset of symptoms of the HIV infection.
In one embodiment, a vaccine composition of the present invention is provided to a subject either before the onset of infection (so as to prevent or attenuate an anticipated infection) or after the initiation of an infection, and thereby protects against viral infection. In some embodiments, a vaccine composition of the present invention is provided to a subject before or after onset of infection, to reduce viral transmission between subjects.
It will be further appreciated that compositions of the present invention can be administered as the sole active pharmaceutical agent, or used in combination with one or more agents to treat or prevent HIV or symptoms associated with HIV infection.
In another embodiments, the present invention provides the composition in, or in the manufacture of a medicament for, the treatment or prevention of HIV infection.
In another embodiment, the present invention provides for use of the composition in, or in the manufacture of a diagnostic agent for, the diagnosis or monitoring of HIV infection or for monitoring an anti HIV treatment protocol.
In some embodiments, the diagnostic agent is an antibody or comprises an antigen binding fragment thereof.
The present invention, an a related embodiment provides, a method of eliciting an immune response in a mammalian subject, the method comprising administering an effective amount of the composition as described herein for a time and under conditions sufficient to elicit an immune response.
Administration of the herein described HIV Env composition or a antibody determined thereby is generally for a time and under conditions sufficient to elicit an immune response comprising the generation of neutralizing antibodies. The immunogenic compositions may be administered in a convenient manner such as by the pulmonary, oral, intravenous (where water soluble), intraperitoneal, intramuscular, subcutaneous, intradermal, intrathecal or suppository routes or implanting (e.g. using slow release formulations). Administration may be systemic or local, although systemic is more convenient. Other contemplated routes of administration are by patch, cellular transfer, implant, sublingually, intraocularly, topically, orally, rectally, vaginally, nasally or transdermally.
As used herein, an “immune response” refers to the reaction of the body as a whole to the presence of a composition of the present invention which includes making antibodies and developing immunity to the composition. Therefore, an immune response to an immunogen also includes the development in a subject of a humoral and/or cellular immune response to the immunogen of interest. A “humoral immune response” is mediated by antibodies produced by plasma cells. A “cellular immune response” is one mediated by T lymphocytes and/or other white blood cells. As used herein, “antibody titres” can be defined as the highest dilution in post-immune sera that resulted in a value greater than that of pre-immune samples for each subject.
The assays for assessing immune responses may comprise in vivo assays, such as assays to measure antibody responses, neutralisation assays and delayed type hypersensitivity responses. In an embodiment, the assay to measure antibody responses primarily may measure B-cell function as well as B-cell/T-cell interactions. For the antibody response assay, antibody titres in the blood may be compared following an antigenic challenge. These levels can be quantitated according to the type of antibody, as for example, IgG, IgG1, IgG2, IgG3, IgG4, IgM, IgA or IgD. Also, the development of immune systems may be assessed by determining levels of antibodies and lymphocytes in the blood without antigenic stimulation. The assays may also comprise in vitro assays. The in vitro assays may comprise determining the ability of cells to divide, or to provide help for other cells to divide, or to release lymophokines and other factors, express markers of activation, and lyse target cells. Lymphocytes in mice and man can be compared in in vitro assays. In an embodiment, the lymphocytes from similar sources such as peripheral blood cells, splenocytes, or lymph node cells, are compared. It is possible, however, to compare lymphocytes from different sources as in the non-limiting example of peripheral blood cells in humans and splenocytes in mice. For the in vitro assay, cells may be purified (e.g., B-cells, T-cells, and macrophages) or left in their natural state (e.g., splenocytes or lymph node cells). Purification may be by any method that gives the desired results. The cells can be tested in vitro for their ability to proliferate using mitogens or specific antigens. The ability of cells to divide in the presence of specific antigens can be determined using a mixed lymphocyte reaction (MLR) assay. Supernatant from the cultured cells can be tested to quantitate the ability of the cells to secrete specific lymphokines. The cells can be removed from culture and tested for their ability to express activation antigens. This can be done by any method that is suitable as in the non-limiting example of using antibodies or ligands which bind to the activation antigen as well as probes that bind the RNA coding for the activation antigen. Also, in an embodiment, phenotypic cell assays can be performed to determine the frequency of certain cell types. Peripheral blood cell counts may be performed to determine the number of lymphocytes or macrophages in the blood. Antibodies can be used to screen peripheral blood lymphocytes to determine the percent of cells expressing a certain antigen as in the non-limiting example of determining CD4 cell counts and CD4/CD8 ratios.
In accordance with these embodiments, the composition is preferably administered for a time and under conditions sufficient to elicit an immune response comprising the generation of Env-specific neutralizing antibodies. The compositions of the present invention may be administered as a single dose or application. Alternatively, the compositions may involve repeat doses or applications, for example the compositions may be administered 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times over relatively long periods in order to enable B-cell maturation and somatic mutation of antibody genes and engender neutralizing antibodies. In some embodiments, the immune response comprises the production of neutralizing antibodies.
In other embodiments, the present invention provides a method of immunising a subject against an HIV infection comprising administering the composition as described herein to the mammalian subject.
In some embodiments, a method of treating or preventing an HIV infection in a mammalian subject is provided, said method comprising administering the composition comprising a remodelled Env immunogen as described herein to the subject for a time and under conditions sufficient to treat an HIV infection in the subject.
Also contemplated are a kit or solid substrate comprising a remodelled Env immunogen or lipid containing particle comprising same as described herein.
The invention provides an Env antigen or a nucleic acid molecule encoding same identified by the process described herein for identifying, producing or selecting modified Env immunogens.
In a further aspect, the present invention provides a process for producing a neutralizing antibody comprising injecting into a subject an immunologically effective amount of the modified Env composition of the present invention, and isolating and purifying the antibody produced. In another embodiment, the present invention provides purified antibodies raised against one or more of the subject HIV Env-based compositions described herein. Preferably, neutralizing antibodies are broadly neutralizing antibodies which neutralize more than one HIV virus from different clades.
Antibodies may be polyclonal or monoclonal. Further, antibodies may be selected for diagnostic, prognostic, therapeutic, prophylactic, and screening purposes typically using criteria known to those of skill in the relevant art. In some embodiments, neutralization potency and cross-clade and cross-Env specificity neutralization ability is tested relative to suitable controls to identify antibodies with superior ability. In other embodiments, cell binding analysis may be performed. Antibodies may be tested on multi-clade pseudovirus panels of hundreds of HIV viruses to assess neutralization breadth and potency. Neutralization breadth may be determined as a percent neutralization with an IC50 or IC90 of less than about 2 ug/ml to less than about 0.1 ug/ml to less than 0.01 ug/ml. Recombinant rescue of monoclonal antibodies may involve the use of B-cell culture systems as described previously. Antibodies may be tested before and after deglycosylation of gp120.
The terms “antibody” and “antibodies” include polyclonal and monoclonal antibodies and all the various forms derived from monoclonal antibodies, including but not limited to full-length antibodies (e.g. having an intact Fc region), antigen-binding fragments, including for example, Fv, Fab, Fab′ and F(ab′)2 fragments; and antibody-derived polypeptides produced using recombinant methods such as single chain antibodies. The terms “antibody” and “antibodies” as used herein also refer to human antibodies produced for example in transgenic animals or through phage display, as well as antibodies, human or humanized antibodies, primatized antibodies or deimmunized antibodies. It also includes other forms of antibodies that may be therapeutically acceptable and antigen-binding fragments thereof, for example single domain antibodies derived from cartilagenous marine animals or Camelidae, or from libraries based on such antibodies. The selection of fragmented or modified forms of the antibodies may also involve consideration of any affect the fragments or modified forms have on the half-lives of the antibody or fragment.
In some embodiments, the antibody is provided with a pharmaceutically or pharmacologically acceptable carrier, diluent or excipient.
In other embodiments, the antibody is selected for diagnosis or prognosis. In some embodiments, kits comprising antibodies determined by the modified Env glycoproteins of the present invention are contemplated.
A “pharmaceutically acceptable carrier and/or a diluent” is a pharmaceutical vehicle comprised of a material that is not otherwise undesirable i.e., it is unlikely to cause a substantial adverse reaction by itself or with the active composition. Carriers may include all solvents, dispersion media, coatings, antibacterial and antifungal agents, agents for adjusting tonicity, increasing or decreasing absorption or clearance rates, buffers for maintaining pH, chelating agents, membrane or barrier crossing agents. A pharmaceutically acceptable salt is a salt that is not otherwise undesirable. The agent or composition comprising the agent may be administered in the form of pharmaceutically acceptable non-toxic salts, such as acid addition salts or metal complexes.
For oral administration, the compositions can be formulated into solid or liquid preparations such as capsules, pills, tablets, lozenges, powders, suspensions or emulsions. In preparing the compositions in oral dosage form, any of the usual pharmaceutical media may be employed, such as, for example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents, suspending agents, and the like in the case of oral liquid preparations (such as, for example, suspensions, elixirs and solutions); or carriers such as starches, sugars, diluents, granulating agents, lubricants, binders, disintegrating agents and the like in the case of oral solid preparations (such as, for example, powders, capsules and tablets). Because of their ease in administration, tablets and capsules represent the most advantageous oral dosage unit form, in which case solid pharmaceutical carriers are obviously employed. Tablets may contain a binder such as tragacanth, corn starch or gelatin; a disintegrating agent, such as alginic acid; and a lubricant, such as magnesium stearate. If desired, tablets may be sugar-coated or enteric-coated by standard techniques. The active composition can be encapsulated to make it stable to passage through the gastrointestinal tract. See for example, International Patent Publication No. WO 96/11698.
For parenteral administration, the composition may be dissolved in a carrier and administered as a solution or a suspension. For transmucosal or transdermal (including patch) delivery, appropriate penetrants known in the art are used for delivering the composition. For inhalation, delivery uses any convenient system such as dry powder aerosol, liquid delivery systems, air jet nebulizers, propellant systems. For example, the formulation can be administered in the form of an aerosol or mist. The compositions may also be delivered in a sustained delivery or sustained release format. For example, biodegradable microspheres or capsules or other polymer configurations capable of sustained delivery can be included in the formulation. Formulations can be modified to alter pharmacokinetics and biodistribution. For a general discussion of pharmacokinetics, see, e.g., Remington's Pharmaceutical Sciences, 1990 (supra). In some embodiments the formulations may be incorporated in lipid monolayers or bilayers such as liposomes or micelles. Targeting therapies known in the art may be used to deliver the agents more specifically to certain types of cells or tissues.
The actual amount of active agent administered and the rate and time-course of administration will depend on the nature and severity of the disease. Prescription of treatment, e.g. decisions on dosage, timing, etc. is within the responsibility of general practitioners or specialists and typically takes into account the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners. Examples of techniques and protocols can be found in Remington's Pharmaceutical Sciences, 1990 (supra).
Sustained-release preparations that may be prepared are particularly convenient for inducing immune responses. Examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the polypeptide, which matrices are in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides, copolymers of L-glutamic acid and ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers, and poly-D-(−)-3-hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods. Liposomes may be used which are of the small (about 200-800 Angstroms) unilamellar type in which the lipid content is greater than about 30% cholesterol, the selected proportion being adjusted for the optimal therapy.
Stabilization of proteins may be achieved by modifying sulfhydryl residues, lyophilizing from acidic solutions, controlling moisture content, using appropriate additives, and developing specific polymer matrix compositions. The in vivo half life of proteins may be extended using techniques known in the art, including, for example, by the attachment of other elements such as polyethyleneglycol (PEG) groups.
Prime-boost immunization strategies as disclosed in the art are contemplated. See for example International Publication No. WO/2003/047617. Thus, compositions may be in the form of a vaccine, vector, DNH priming or boosting agent.
In some embodiments, kits comprising the herein described modified Enc glycoprotein are conveniently used for (or are for use in) diagnosis or prognosis of viral infection, or pathogen monitoring or serosurveillance kits, and optionally include packaging, instructions and various other components such as buffers, substrates, antibodies or ligands, control antibodies or ligands, and detection reagents.
The term “isolated” and “purified” means material that is substantially or essentially free from components that normally accompany it in its native state. For example, an “isolated nucleic acid molecule” refers to a nucleic acid or polynucleotide, isolated from the sequences which flank it in a naturally-occurring state, e.g., a DNA fragment which has been removed from the sequences that are normally adjacent to the fragment. In particular, an isolated Env includes in vitro isolation and/or purification of a protein from its natural cellular environment, or from association with other components of a cell. Without limitation, an isolated nucleic acid, polynucleotide, peptide, or polypeptide can refer to a native sequence that is isolated by purification or to a sequence that is produced by recombinant or synthetic means.
Reference to variants includes mutations, parts, derivatives and functional analogs. While the mutations described in the Examples were selected for by forced evolution, further mutants may be included using an iterative approach. In designing amino acid sequence mutants, the location of the mutation site and the nature of the mutation will depend on characteristic(s) to be modified. The sites for mutation can be modified individually or in series, e.g., by (1) substituting first with conservative amino acid choices and then with more radical selections depending upon the results achieved, (2) deleting the target residue, or (3) inserting other residues adjacent to the located site. Amino acid sequence deletions generally range from about 1 to 15 residues, more preferably about 1 to 10 residues and typically about 1 to 4, 1 to 5 or 1 to 10, or 1 to 15 or 1 to 20 contiguous amino acid residues. Substitution mutants have at least one amino acid residue in the polypeptide molecule removed and a different residue inserted in its place. The sites of greatest interest for substitutional mutagenesis have been identified herein as the DSR, MPER and the V1 regions. Glycosylation variants can be assessed by known methods such as by mass spectrometry.
The present invention is further described by the following non-limiting Examples.
The CMV promoter-driven HIV-1 AD8 Env expression vector, pCDNA3.1-AD8env, is described elsewhere [33]. pΔKAD8env was derived by religation of the end-filled HindIII and EcoRI sites of pCDNA3.1-AD8env. Mutants of the pAD8 infectious clone (obtained from K. Peden [70]) were prepared by transferring the EcoRI-BspMI env-containing fragment from pCDNA3.1-AD8env vectors into pAD8. In vitro mutagenesis of the gp41 region was carried out using the Quikchange protocol (Stratagene).
Virus stocks were prepared by transfecting 293T cell monolayers with pAD8 infectious clones using Fugene 6 or Fugene HD (Roche). Virus-containing transfection supernatants were normalized according to reverse transcriptase (RT) activity, and then used to infect U87.CD4.CCR5 astroglioma cells (from H. Deng and D. Littman [71], NIH AIDS Research and Reference Reagent Program) in 25 cm2 culture flasks. The supernatants were assayed for RT activity at various time points. To assess the transmission of cell-associated viruses, HIV-1 particles were pseudotyped with VSV G by cotransfection of 293T cells with pAD8 and pHEF-VSV G (from Dr. L.-J. Chang [72] NIH AIDS Research and Reference Reagent Program). U87.CD4.CCR5 cells in 25 cm2 culture flasks were inoculated with the HIV-VSV G pseudotypes, and then, at 24-h postinfection, trypsinized to remove surface-adsorbed virions. The cells were replated and then cultured for 10 days. The culture supernatants were assayed for RT activity at days 3, 7 and 10. For long-term cultures of viral mutants, the day-10 cell-free culture supernatants were filtered (0.45 μm pore size) and normalized according to RT activity prior to the next passage (5 passages in total). Genomic DNA was extracted from infected cells using Qiagen DNeasy Blood and Tissue kit. The viral DNA fragment encompassed by nucleotides 5954-9096 (HIV-1HXB2R numbering convention) was PCR-amplified using Expand HiFi (Roche) and the primers, 5′-GGCTTAGGCATCTCCTATGGCAGGAAGAA, SEQ ID NO:1, (Env1A) and 5′-TAGCCCTTCCAGTCCCCCCTTTTCTTTTA, SEQ ID NO:2, (Env1M) [73]. The amplified sequences were ligated into pΔKAD8env (KpnI-XbaI) and the entire env open reading frame sequenced using ABI BigDye terminator v3.1.
Single cycle infectivity assays were conducted as described [24]. Env-pseudotyped luciferase reporter viruses were produced by cotransfecting 293T cells with pΔKAD8env plus the luciferase reporter virus vector, pNL4.3.Luc.R−E− (NIH AIDS Research and Reference Reagent Program, from N. Landau [74]), using Fugene HD. The infectivity of pseudotyped viruses was determined in U87.CD4.CCR5 cells using the Promega luciferase assay system at 48 h postinfection.
Twenty four h after transfection with pΔKAD8env vectors, 293T cells were lysed for 10 min on ice in PBS containing 1% Triton X-100, 0.02% sodium azide, 1 mM EDTA. The lysates were clarified by centrifugation for 10 min at 10,000×g at 4° C. prior to SDS-PAGE under reducing conditions. The proteins were transferred to nitrocellulose and blotted with antibodies C8 to gp41 [75] and DV-012 to gp120 [76] (from G. Lewis and M. Phelan, respectively, NIH AIDS Research and Reference Reagent Program). The immunoblots were developed with Alexa Fluor 680-conjugated goat anti-mouse or donkey anti-sheep immunoglobulin (Invitrogen) and scanned in a LI-COR Odyssey infrared imager. For virion analysis, supernatants from pAD8-transfected 293T cells were centrifuged over 1.5 ml 25% w/v sucrose/PBS cushions (Beckman SW41 Ti rotor, 25,000 rpm, 2.5 h, 4° C.) prior to reducing SDS-PAGE and western blotting with DV-012 to detect gp120 and pooled IgG from HIV-1-infected individuals to detect Gag proteins.
293T cells were transfected with pΔKAD8env vectors. At 24-h post transfection, the cells were incubated for 30 min in cysteine and methionine-deficient medium (MP Biomedicals), and then labelled for 45 min with 150 μCi Tran-35S-label (MP Biomedicals). The cells were washed and then chased in complete medium for 5 h prior to lysis. Cell lysates and clarified culture supernatants were immunoprecipitated with pooled IgG from HIV-1-infected persons and protein G Sepharose and subjected to SDS-PAGE in the presence of β-mercaptoethanol. The labelled proteins were visualized by scanning in a Fuji phosphorimager.
Cell-cell fusion assays were conducted as previously described [24]. Briefly, 293T cells were cotransfected with pΔKAD8env and the bacteriophage T7 RNA polymerase expression vector, pCAG-T7 [77]. BHK21 target cells were cotransfected with pc.CCR5 (AIDS Research and Reference Reagent Program from N. Landau [78]) and pT4luc, a bicistronic vector that expresses human CD4 from a CMV promoter and firefly luciferase from a T7 promoter [13]. At 24 h posttransfection, targets and effectors were cocultured in triplicate in a 96-well plate (18 h, 37° C.) and then assayed for luciferase activity (SteadyGlo, Promega).
Purified IgG of brNAbs 2F5 [63], 4E10 [64] and IgGb12 [79, 80] were obtained from Polymun Scientific, while the HR2 peptide analogue, C34 [40], was purchased from Genscript. Neutralization assays were conducted using TZM-bl cells (obtained from J. C. Kappes, X. Wu and Tranzyme Inc., NIH AIDS Research and Reference Reagent Program [81-83]), a HeLa cell line expressing CD4 and CCR5 and harbouring integrated copies of the luciferase and β-galactosidase genes under control of the HIV-1 promoter. Virus stocks produced by pAD8-transfected 293T cells and determined to give ˜1.5×106 relative light units (RLU) following infection of TZM-bl cells, were mixed with an equal volume of serially diluted IgG or C34 peptide and incubated for 1H at 37° C. One hundred μl of the virus-IgG mixture was then added to TZM-bl cells (104 cells in 100 μl per well of a 96-well tissue culture plate) and incubated for 2 days prior to lysis and assay for luciferase activity (Promega, Madison, Wis.). For experiments with the CCR5 antagonist maraviroc (NIH AIDS Research and Reference Reagent Program [84]), the cells were preincubated for 1 h at 37° C. with the drug prior to incubation with virus for 48 h. Neutralizing activities were measured in triplicate and reported as the average percent luciferase activity.
The gp120-gp41 association phenotype of the WL/KD mutant was investigated by immunoprecipitation of biosynthetically labelled Env glycoproteins expressed in 293T cells [33]. The WL/KD mutation led to >95% of total gp120 being sloughed into the culture supernatant (
Long-Term Culture of HIV-1AD8-WL/KD.
Viruses derived from 2 independent HIV-1AD8 proviral clones carrying WL/KD were subjected to long-term culture in U87.CD4.CCR5 cells with serial passaging of cell-free virus onto fresh cells every 10 days. Evidence of replication was not observed for either clone, even after 50 days of culture (
The env region was PCR-amplified from genomic DNA isolated at days 10, 20, 30, 40 and 50, the PCR products were cloned into pΔKAD8env, and the entire env region was sequenced. WLKD-1. A D601H pseudoreversion emerged at day 10 (2/6 clones, WL/KH) prior to the appearance of D674E in the MPER at day 20, which persisted throughout the culture period. The genotypes observed over the 50-day culture period included WL/KH (9/35 clones), W596L/K601H/D674E (WL/KH/DE [10/35 clones]), L85M/W596L/K601H/D674E (LM/WL/KH/DE [6/35 clones]), W596L/K601H/D674G (WL/KH/DG [4/35 clones]), L85M/W596L/K601H (LM/WL/KH [1/35 clones]), and W596L/K601H/D674N (WL/KH/DN [1/35 clones]) (
The dominant genotypes were reconstructed in the context of the pAD8 proviral clone. In the case of WLKD-1, cell-free virus-initiated replication in U87.CD4.CCR5 cells was partially restored by D601H in the DSR (WL/KH) and was optimised further by D674E in the MPER (WL/KH/DE) (
The infectivity associated with revertant genotypes was further examined in a single cycle infectivity assay employing Env-pseudotyped luciferase reporter viruses. The infectivity of WL/KD for U87.CD4.CCR5 cellular targets was reduced by ˜2.5 log10 with respect to WT (
It was investigated whether the modulation of infectivity by D674E, D674N and D674G occurred via a functional link to Leu-596 and His-601 or whether it could be explained by a generalized enhancement or inhibition in Env function.
The functional advantages conferred by D601H and D674E were less obvious in the single cycle infectivity assay when compared to 14-day replication experiments (compare
The synthesis and processing of the cloned Env glycoproteins were examined by western blot. The gp120-specific polyclonal antibody, DV012, (
The membrane fusion activities of selected revertant Env sequences were examined in a cell-to-cell fusion assay. In this context, Env is expressed in the absence of other viral proteins and is therefore not subjected to the conformational constraints that may be imposed by matrix-gp41 cytoplasmic tail interactions present in virus [36-39]. The assay was conducted at limiting Env concentrations (0.25 μg pΔKADenv) to enable detection of subtle changes in fusion function. Consistent with the cell-free virus infectivity data, WL/KD blocked cell-cell fusion, WL/KH exhibited partially restored fusion function and D674N and D674G mutations were inhibitory in a WL/KH context (
To determine whether WL/KH and WL/KH/DE are associated with structural changes in gp41, neutralizing agents were used to probe functional virion-associated gp120-gp41 complexes. Virus stocks, produced by transfecting 293T cells with pAD8 infectious clones, were adjusted to produce ˜1.5×106 RLU following 48 h of infection of TZM-bl cells. The viruses were then incubated with the neutralizing agents for 1 h prior to infection of naïve TZM-b1 cells. In the case of maraviroc, target cells were pretreated with the CCR5 antagonist for 1 h prior to infection. The CD4 binding site brNAb, IgGb12, and the CCR5 antagonist, maraviroc, neutralized WT, WL/KH and WL/KH/DE to similar extents (the maraviroc IC50 and IC90 values for WT were not significantly different to those obtained with the mutants) indicating that gp120-CD4-CCR5 interactions had not been affected by the mutations in gp41 (
The forced evolution of WL/KD mutant viruses with severely disrupted gp120-gp41 association led to the emergence of replication-competent revertants containing a D601H pseudoreversion in the DSR plus D674E or D674N 2nd site mutations in the MPER. In the case of WLKD-2 clones the ΔT394-W395 deletion in V4 was also observed. The WL/KH and WL/KH/DE viruses exhibited greater sensitivity to the brNAbs, 2F5 and 4E10, indicating that the restoration of function was associated with structural changes in Env that increase the accessibility of neutralization epitopes within the MPER. Our data reveal a functional linkage between the DSR and MPER of gp41 and a novel approach for improving the accessibility of conserved neutralization epitopes within the MPER in a virion context.
The severe shedding phenotype of WL/KD is likely to have resulted from the combined effects of decreased hydrophobic side chain bulk and an additional negative charge in the contact site that destabilizes gp120-gp41 association. Phenotypic analysis of the revertant genotypes indicated that D601H is a key evolutionary step that partially restores gp120-gp41 association. Histidine at 601 introduces animidazole moiety into the association site, which would partially compensate for the loss of the indole ring of Trp-596 and removes the negative charge contributed by Asp-601. Interestingly, Leu-596 was maintained in both long-term cultures, indicating that the smaller hydrophobic sidechain is preferred at this position when His is present at 601. The combination of His-601 with D674E led to improved single-cycle and multi-cycle cell-free virus-initiated infectivity without detectable further improvement in gp120-gp41 association. The MPER mutation therefore appears to act at the level of virus entry. The modelling of this change into the 3D structure of an MPER peptide determined by NMR (PDB entry, 2PV6 [25]) suggests that the compensatory nature of D674E is related to MPER flexibility. In membranes, the MPER comprises an N-terminal helix connected to a C-terminal helix via a hinge composed of Phe-673, which is buried in the lipid phase, and a polar residue at position 674, which is solvent-exposed (
In contrast to the data obtained with cell-free virions, replication levels at (WL/KH, WL/KH/DN, ΔTW/WL/KH/DN) or better (WL/KH/DE) than those of WT were achieved by the revertants when infections were initiated with VSV G pseudotyped viruses. In this latter system, the initial infection rounds will be largely mediated by the highly fusogenic VSV G present in the viral envelope thereby normalizing WT and mutant virus infectivity, while subsequent infection rounds will be mediated by gp120-gp41 present on virions transmitted directly from cell to cell via virological synapses, in addition to nascent cell-free virions. Direct cell-cell viral transmission has been calculated to be at least 8-times more efficient than cell-free viral spread and is believed to be due to higher effective multiplicity of infection and virus viability within virological synapses [35]. It may be that in the case of cell-free virions, which encounter receptors following solution-phase diffusion, gp120 is shed from unstable WL/KH, WL/KH/DE and WL/KH/DN gp120-gp41 complexes during the lag time between budding and attachment, thereby decreasing infectivity over time. By contrast, in directed viral transmission across virological synapses, budding, receptor binding, virion maturation and entry appear to be closely linked and to occur over short timeframes [34, 42, 43], which may limit the loss of gp120 from virions prior to receptor encounter. Dale et al. [42] recently reported that in cell-cell viral transmission, receptor attachment is mediated by immature virions, and Env activation for fusion occurs later following viral maturation in the endosome. This contrasts cell-free virus infection where receptor binding is largely mediated by mature virions. It may be that the reverting mutations act optimally in the context of a virion gp120-gp41 complex maintained in an inactive form through interactions between the gp41 cytoplasmic domain and immature Gag [37, 39], prior to receptor engagement and activation for fusion. Overall, these data suggest that the cell-cell mode of viral spread plays the key role in the mechanism of reversion. This idea is consistent with the finding that the WL/KH/DN and ΔTW/WL/KH/DN env genotypes coexist with WL/KH in the WLKD-2 culture even though the former exhibited lower cell-free virus infectivity. It is interesting that the boosts to infectivity associated with the addition of D674E to W596L/K601H did not correlate with enhanced cell-cell fusion activity, where the cell-surface expressed glycoproteins function independently of other virion components. These data are consistent with a Leu-596-His-601-Glu-674 functional interaction that is dependent on virion assembly and the structural constraints imposed on the Envectodomain, including the MPER, by Gag-gp41 cytoplasmic tail interactions [36-39].
Early mutational studies indicated that the DSR is associated with the C1 and C5 regions of gp120 [11-13, 33], which project from the base of gp120 [17] (
The WL/KH (and WL/KH/DE) mutations were associated with sensitivity to the 2F5 and 4E10 MPER-directed brNAbs, indicating a structural change in Env that increases MPER accessibility. Two neutralization mechanisms have been proposed for 2F5 and 4E10: i) direct interaction with the MPER in neutralization-sensitive viral strains; ii) Env-receptor interaction-triggered MPER accessibility to brNAb in resistant strains [46]. We have found that the macrophage-adapted AD8 strain is relatively resistant to these brNAb suggesting that the MPER is sterically occluded in the WT viral AD8 Env complex and the WL/KH gp120-gp41 association site mutation leads to a more open Env structure, enabling better epitope access for 2F5 and 4E10. Alternatively or additionally, WL/KH may be associated with structural change in the MPER itself, within creased MPER flexibility and/or altered membrane interactions facilitating paratope-mediated extraction of the 2F5 and 4E10 epitopes from the envelope. We previously reported that the contributions of Trp-596 and Lys-601 to gp120-gp41 association and membrane fusion are influenced by sequence changes in V1, V2, and V3 [33], which are predominantly associated with the evolution of neutralization resistance [50-56], as well as coreceptor preference and cellular tropism [57-60]. Thus the gp120-gp41 association site appears structurally and functionally adaptable, perhaps to maintain glycoprotein function during gp120-gp41 evolution. The observation here of functional crosstalk between the DSR and MPER implies that the structural adaptation of the gp120-gp41 synapse in order to cope with the evolution of other glycoprotein domains is also linked to changes in MPER structure that alters the ability of conserved neutralization epitopes therein to be bound by antibody.
MPER-specific brNAbs are of particular interest to the HIV-1 vaccine field due to the conserved nature of their epitopes and their neutralization breadth [20, 61-64]. Biophysical and structural studies have indicated that membrane-anchored MPER conformations are optimally bound by 2F5 and 4E10-like brNAbs [65], however the goal of developing a vaccine that presents the MPER in a lipid environment and produces high-titre 2F5- and 4E10-like brNAbs has not yet been realized [32, 66-69]. The data presented here indicate that changes to the gp120-gp41 association site can increase the availability of the 2F5 and 4E10 epitopes in virus and point to a new approach for improving the accessibility of MPER epitopes in virion-based immunogens.
The preparation of the cytomegalovirus promoter-driven HIV-1AD8 Env expression vector, pCDNA3.1-AD8env, is described elsewhere [28]. pΔKAD8env was derived by religation of the end-filled HindIII and EcoRI sites of pCDNA3.1-AD8env. In vitro mutagenesis of the gp41 region was carried out by overlap extension PCR. Mutants of the pAD8 infectious clone (obtained from K. Peden [89] were prepared by transferring the EcoRI-BspMI env-containing fragment from pCDNA3.1-AD8env vectors into pAD8. Bacteriophage T7 promoter-driven gp120 expression vectors, based on pTM.1 [90], were generated by ligating PCR-amplified HIV-1AD8 gp120 fragments into the NdeI and StuI sites of pTMenv.2 [91] to give pTM-AD8gp120.
PBMC infections were conducted as described previously [92]. Briefly, PBMCs isolated from buffy packs (Red Cross Blood Bank, Melbourne) were stimulated with phytohemagglutinin (10 μg/ml; Murex Diagnostics) for 3 days in RPMI 1640 medium containing 10% fetal calf serum and interleukin-2 (10 units/ml; Boehringer-Mannheim). Virus stocks were prepared by transfecting 293T cell monolayers with pAD8 infectious clones using Fugene 6 (Roche). Virus-containing transfection supernatants were normalized according to reverse transcriptase (RT) activity, and then used to infect 105 PBMCs in a 96-well tissue culture plate (eight 10-fold serial dilutions of each virus were tested in triplicate). The supernatants were assayed for RT activity at various time points.
Phytohemagglutinin-stimulated PBMCs were infected with equivalent amounts of wild type (WT) and K601D-mutated HIV-1AD8 (according to RT activity) in parallel and maintained in culture for 10 days. Cell-free culture supernatants were filtered (0.45 μm pore size) and normalized according to RT activity prior to the next passage (5 passages in total). Genomic DNA was extracted from infected PBMCs using Qiagen DNeasy. The viral DNA fragment encompassed by nucleotides 5954-9096 (HXB2R numbering convention) was PCR-amplified using Expand HiFi (Roche) and the primers, 5′-GGCTTAGGCATCTCCTATGGCAGGAAGAA, SEQ ID NO:1, (Env1A) and 5′-TAGCCCTTCCAGTCCCCCCTTTTCTTTTA, SEQ ID NO:2, (Env1M) [93]. The amplified sequences were ligated into pGEM-T or pΔKAD8env (KpnI-XbaI) and the entire env open reading frame sequenced using ABI BigDye terminator 3.1.
Lysates of Env-expressing 293T cells or virions pelleted from pAD8-transfected 293T cell supernatants were subjected to SDS-PAGE under reducing conditions, transferred to nitrocellulose and then probed with mAb C8 to gp41 (from G. Lewis [94], DV-012 to gp120 (from M. Phelan [95,96], or mAb 183 to CA (from B. Chesebro and K. Wehrly [97,98](AIDS Research and Reference Reagent Program, NIAID) as described [28].
Cell-cell fusion assays were conducted as described [76]. Briefly, 293T effector cells were cotransfected with pCDNA3.1-AD8env or pΔKAD8env and pCAG-T7 [99] plasmids, while BHK21 target cells were cotransfected with pT4luc [27] and pc.CCR5 (AIDS Research and Reference Reagent Program from N. Landau [100] or a panel of CCR5 mutants in the pcDNA3 expression vector (kind gifts of J. S. Sodroski and R. W. Doms [101,102]). The Y14N mutation was introduced to pc.CCR5 using the Quikchange II XL kit (Stratagene). At 24 h posttransfection, targets and effectors were cocultured in triplicate in a 96-well plate (18 h, 37° C.) and then assayed for luciferase activity (Promega SteadyGlo, Madison, Wis.). The sensitivities of WT and mutant Env proteins to the fusion inhibitor peptide C34 (WMEWDREINNYTSLIHSLIEESQNQQEKNEQELL, SEQ ID NO:3; Mimotopes, Australia [57]) were determined by coculturing effector and target cells in the presence of serially diluted C34. The sensitivities of WT and mutant Env proteins to sCD4 (NIH AIDS Research and Reference Reagent Program) were determined by incubating the Env-expressing 293T cells with a dilution series of sCD4 for 3.5 h followed by coculturing the effector and target cells in the presence of sCD4 for 8 h.
Single cycle infectivity assays were conducted as described [76]. Briefly, Env-pseudotyped luciferase reporter viruses were produced by cotransfecting 293T cells with pCDNA3.1-AD8env or pΔKAD8env vectors plus the luciferase reporter virus vector, pNL4.3.Luc.R−E− (AIDS Research and Reference Reagent Program, N. Landau [103]), using Fugene 6. The infectivity of pseudotyped viruses was determined in U87.CD4.CCR5 cells (AIDS Research and Reference Reagent Program, H. Deng and D. Littman[104]).
293T cells were transfected with pCDNA3.1-AD8env or pΔKAD8env vectors. At 24-h posttransfection, the cells were incubated for 30 min in cysteine and methionine-deficient medium (MP Biomedicals, Seven Hills, NSW, Australia), and then labelled for 45 min with 150 μCi Tran-35S-label (MP Biomedicals). The cells were then washed and chased in complete medium for 5-6 h prior to lysis. Cell lysates and clarified culture supernatants were immunoprecipitated with IgG14 or HIVIG and protein G Sepharose and subjected to SDS-PAGE in the presence of β-mercaptoethanol [28]. The labelled proteins were visualized by scanning in a Fuji phosphorimager. Quantitation of bands was performed using Image Gauge (FUJIFILM) software.
gp120-sCD4 Binding Assay
293T cells were cotransfected with pTM-AD8gp120 and pCAG-T7 vectors using Fugene 6. At 24-h posttransfection, the cells were incubated for 30 min in cysteine and methionine-deficient medium, labelled for 45 min with 150 μCi Tran-35S-label, and then washed and chased in complete medium for 6 h. The clarified culture supernatants were adjusted to 0.6 M KCl, 1 mM EDTA and 1% w/v Triton-X100 and the gp120 content quantified following immunoprecipitation with IgG14 and protein G Sepharose, reducing SDS-PAGE and scanning in a Fuji phosphorimager. Equivalent amounts of WT and mutant gp120 proteins were incubated with serial dilutions of sCD4 (1 h, room temperature) and then incubated with monoclonal antibody (mAb) OKT4 and BSA-Sepharose on a vibrating platform (30 min, room temperature). After pelleting the BSA-Sepharose, protein complexes were coprecipitated using protein G-Sepharose and gp120 quantified following reducing SDS-PAGE and scanning in a Fuji phosphorimager.
Neutralization assays were conducted using a modification of the method of Dhillon et al. [105]. Briefly, a solution of Env-pseudotyped luciferase reporter viruses determined previously to give 5×105 relative light units (RLU) following infection of U87.CD4.CCR5 cells was mixed with an equal volume of serially diluted IgG and incubated for 1 h at 37° C. One hundred microliters of the virus-IgG mixtures was then added to U87.CD4.CCR5 cells (104 cells per well of a 96-well tissue culture plate, 100 microlitres) and incubated for 2 days prior to lysis and assay for luciferase activity (Promega, Madison, Wis.). Neutralizing activities for antibody samples were measured in triplicate and reported as the average percent luciferase activity. Purified IgG of monoclonal NAbs 2F5, 2G12 and IgGb12 were obtained from Polymun Scientific (Austria), PG16 was obtained from the International AIDS Vaccine Initiative, while HIVIG was obtained from F. Prince through the NIAID AIDS Research and Reference Reagent Program.
A homology-based model of HIV-1AD8 V1V2 was generated from PDB entry 3U4E [7] using the Modeller algorithm [106] within Discovery Studio, version 3.0 (Accelrys). Five independent models were generated from iterative cycles of conjugate-gradient energy minimization against spatial constraints derived from the template crystal structure. The model with the lowest energy (probability density function) was glycosylated in silico with oligomannose side chains using the glycosciences.de server [107,108].
The gp120-gp41 association defective DSR mutant HIV-1AD8-K601D was subjected to serial PBMC passage in order to select suppressor mutations. It was reasoned that the locations of suppressors would point to structural elements that are functionally linked to the DSR, thereby shedding light on its role in Env function. It was first confirmed that the K601D mutation promoted the shedding of gp120 into the culture supernatant of 293T cells transfected with pcDNA3.1-AD8env, as determined by radio immunoprecipitation (
In order to identify suppressors of K601D, the env region was PCR-amplified from genomic DNA isolated from infected cells at days 20, 30, 40 and 50 for culture P0, and days 30 and 50 for culture P2. The PCR products were cloned into the pΔKAD8env expression vector for DNA sequencing. The K601D mutation was retained in P0 clones isolated at days 20 and 30 (
In an independent PBMC long-term culture (P2), the replication competence of the mutant virus approached that of WT by the 3rd passage (
To determine how replication was restored to the K601D virus in the long-term PBMC cultures, we reconstructed the dominant P0 and P2 genotypes in the context of the pAD8 proviral clone and examined viral replication in two independent PBMC donors. Both P0 mutations, L494I within C5 and K601N within the DSR, partially restored replication in the PBMCs of one donor but not in the second, apparently less permissive donor (
The pΔKAD8env expression vector was next employed to further dissect the functional linkages between position 601 within the DSR, the Asn136 and Asn141/142PNGS mutations in V1 and the L494I mutation in C5 in gp120-gp41 association and cell-cell fusion assays. The results presented in
The ΔN139INN/K601D and ΔN139INN/K601N P2 clones exhibited 74-77% of WT fusion activity (P<0.05) with only marginal restoration of gp120-gp41 association, even though only the latter clone was competent for replication in PBMCs (
It was next asked whether the V1 PNGS mutations restored function via a functional link to Asp or Asn at position 601 in the DSR or whether a generalized enhancement in Env function could explain the restored phenotypes.
Finally, it was asked if the V1 PNGS mutations at 136 and 142 restored function via a specific link to position 601 in the DSR, or whether deletion of any of the 6 PNGSs in V1V2 of the AD8 strain (
The effects of T138N, L494I and ΔN139INN on CD4 binding ability, sensitivity to sCD4 inhibition, CCR5 utilization and sensitivity to the C34 fusion HR2-based inhibitor peptide were examined in order to further elucidate the mechanism whereby function was restored in the context of a mutated DSR. CD4 binding curves were generated by incubating a constant amount of biosynthetically labelled WT and mutated gp120 with serial dilutions of sCD4, coimmunoprecipitation of gp120-sCD4 complexes with mAb OKT4, followed by SDS-PAGE and densitometry of gp120 bands. Similar CD4 binding curves were observed for WT, T138N/L494I and ΔN139INN gp120 mutants (
The abilities of T138N/L494I/K601N, ΔN139INN/K601N and WT Envs to utilize a panel of CCR5 coreceptor mutants were next compared in order to determine if alterations to the mode of CCR5 engagement could account for reversion. The results presented in
It was considered that an increase in the efficiency of a post receptor-binding event, such as 6-helix bundle formation, could aid in the functional compensation of the DSR mutations. We therefore asked if the mutations in gp120 and K601N led to changes in sensitivity to the HR2 synthetic peptide analogue, C34, which blocks fusion by binding to the coiled coil of HR1 helices in a receptor-triggered prehairpin intermediate conformation of gp41 [17,57]. It was expected that faster 6-helix bundle folding kinetics would correspond to decreased C34 sensitivity.
The DSR mediates association with gp120 and may play a role in the activation of gp41 by responding to receptor-induced changes in gp120 [27,28,29,30,31]. To better understand how glycosylation in V1 impacts on gp120-gp41 interactions, the functional effects of T138N, L494I, T138N/L494I and ΔN139INN mutations on two other gp120 contact residues within the DSR, Leu593 and Trp596, in addition to Lys601 were assessed [27,28]. While L593V and K601D mutations resulted in decreased gp120-gp41 association and cell-cell fusion function, the W596L mutant exhibited WT levels of gp120-gp41 association but reduced cell-cell fusion by ˜40% at subsaturating Env (P<0.02, 2 sample t-test, unequal variances) (
It was therefore asked whether the T138N and ΔN139INN mutations were linked to changes in the neutralization sensitivity of Env-pseudotyped reporter viruses. The results (
A salient feature of HIV-1 Env is its rapid acquisition of mutations at PNGSs that enable evasion of the adaptive immune response, while maintaining key functions such as gp120-gp41 association, receptor recognition and membrane fusion. Here it was found that glycan changes in V1 that are associated with neutralization sensitivity are linked to a remodelling of the gp120-gp41 association site. It is proposed that this represents a mechanism for the functional adaptation of the highly conserved gp120-gp41 association site to an evolving glycan shield in a setting of neutralizing antibody selection.
The gp120 shedding phenotype of K601D (and K601N) emphasises the importance of the Cys598-Ser-Gly-Lys-Leu-Ile-Cys604 loop segment of the DSR in gp120-gp41 association. Lys601 is conserved in HIV-1 isolates (
X-ray crystallography has indicated that V1V2 comprises a conserved 4-stranded β-sheet minidomain that is stabilized by 2 interstrand disulfide bonds (Cys126-Cys196 and Cys131-Cys157). The highly variable segments and PNGSs of V1V2 are for the most part contained within the connecting loops (
A mechanism for suppression of the K601D fusion phenotype was indicated by an analysis of combination mutants comprising T138N, L494I and ΔN139INN with various DSR mutations known to affect glycoprotein association and/or fusion function [27,28]. The T138N/L494I combination rendered fusion function largely independent of the DSR residues Leu593 and Lys601, whereas less dependence on Lys601 was observed with ΔN139INN. The boosts in fusion function provided to L593V and K601D by these V1 and C5 mutations generally correlated with the restoration of gp120-gp41 association. In the case of W596L, the T138N and ΔN139INN glycan mutations were sufficient to confer WT fusion levels, but this did not involve changes to the WT-like gp120-gp41 association phenotype of W596L. The fusion gains conferred to W596L by T138N and ΔN139INN may involve transduction of a receptor-induced activation signal from gp120 to gp41 through the association site in manner that is less dependent on Trp596. The earlier finding that W596L blocks sCD4-induced formation of the gp41 prehairpin intermediate indicates that Trp596 can play a role in receptor-triggered gp41 activation [29]. Overall, these data suggest that the glycan changes observed here involve a structural remodeling of the gp120-gp41 association interface such that Env function is less dependent on particular gp120-contact residues within the DSR, e.g. Trp596 [27,28]. The maintenance of a functional Env complex in these cases may involve alternative inter-subunit contacts mediated by other DSR residues implicated in gp120 association [e.g. Gln591, Gly597, Thr606 and Trp610 [27,28,75]], and/or other regions of gp41 that appear to interact with gp120, including the fusion-peptide proximal segment [76], HR1 [77,78], and HR2 [75]. Interestingly, it was observed that the C34 HR2 peptide analog inhibited fusion mediated by WT, T138N/L494I/K601N and ΔN139INN/K601N Env to the same extent, indicating that the prefusion gp41-to-6-helix bundle refolding process was not affected by these amino acid changes in gp120 and gp41.
The V1V2 mini domain has been shown to modulate the accessibility and/or conformation of the CD4-binding site and V3 loop [38,79,80,81,82], the latter of which mediates chemokine receptor binding. However, our data argue against a reversion mechanism involving altered gp120-receptor interactions since the sCD4-binding abilities of gp120 molecules bearing 2nd and 3rd site mutations in various combinations were not significantly different to the WT. Furthermore, the T138N/L494I/K601N and ΔN139INN/K601N mutants were able to mediate WT levels of fusion with targets expressing various CCR5 mutants, including the low affinity Y14N coreceptor, thereby also ruling out altered gp120-CCR5 interactions as a potential reversion mechanism. It should however be noted that ΔN139INN/K601N-mediated fusion appeared to be subtly more resistant to sCD4 inhibition in comparison to WT and T138N/L494I/K601N. ΔN139INN/K601N exhibits weaker gp120-gp41 association than WT and T138N/L494I/K601N despite near-WT replication and fusion competence and WT-like sCD4 binding characteristics for soluble gp120 containing ΔN139INN. sCD4 has been shown to primarily inhibit HIV-1 infection by inducing a transiently activated glycoprotein complex that rapidly undergoes irreversible conformational changes linked to a loss of function [83]. This sCD4-mediated inactivation correlates with a rapid decay in exposure of the HR1 coiled coil groove of the fusion-activated gp41 prehairpin intermediate. It may be that the apparently looser association between gp120 and gp41 of ΔN139INN/K601N alters the conformational pathway of the sCD4-induced state to enable maintenance of fusion competence. Overall, these data lend support to the idea that the changes in V1 specifically impact on the gp120-gp41 association site.
A striking feature of gp120-gp41 is the occlusion of the conserved protein surface by a glycan shield comprising ˜24 N-linked glycans on gp120 and 4-5 on gp41 [3,84,85,86,87]. An evolving glycan shield is an important mediator of viral escape from NAbs where an increase in the number and/or a change in the position of PNGSsalter NAb sensitivity [35]. A number of studies have indicated that V1V2 plays a particularly important role in regulating neutralization resistance [36,38,41,42], which generally correlates with V1V2 elongation and insertion of PNGSs, in some cases at and C-terminal to position 136 (
As shown herein, changes at the 136 and 142 V1 glycans that are associated with neutralization sensitivity appear to remodel the gp120-gp41 association site, rendering certain highly conserved gp120-contact residues (i.e. Leu593, Trp596 and Lys601) less important for gp120 association and fusion function, and thereby implying that alternative gp120-gp41 contact residues become utilized for these functions. The allosteric modulation of the conserved DSR-C1-05 synapse by distal V1 glycans may represent a mechanism whereby functionally relevant gp120-gp41 association is maintained as the virus acquires neutralization resistance due to the evolution of its glycan shield.
The cytomegalovirus promoter-driven HIV-1AD8Env expression vector, pΔKAD8env, is described above, as are the ΔN139INN- and W596L/K601H/D674E mutated versions. The ΔN139INN and W596L/K601H/D674E mutations were combined in a single pΔKAD8env vector by replacing the PpuMI DNA fragment of pΔKAD8env-ΔN139INN with that of pΔKAD8env-WL/KH/DE to give pΔKAD8env-ΔN139INN/WL/KH/DE, referred to as ΔNINN/WL/KH/DE (Figure. 18).
Env-pseudotyped luciferase reporter viruses were produced by cotransfecting 293T cells with pΔKAD8env vectors plus the luciferase reporter virus vector, pNL4.3.Luc.R−E− using Fugene 6. The infectivity of pseudotyped viruses was determined in U87.CD4.CCR5 cells.
A solution of Env-pseudotyped luciferase reporter viruses determined previously to give 1-2×105 relative light units (RLU) following infection of U87.CD4.CCR5 cells was mixed with an equal volume of serially diluted IgG and incubated for 1 h at 37° C. One hundred microliters of the virus-IgG mixtures was then added to U87.CD4.CCR5 cells (104 cells per well of a 96-well tissue culture plate, 100 microlitres) and incubated for 2 days prior to lysis and assay for luciferase activity (Promega, Madison, Wis.). Neutralizing activities for antibody samples were measured in triplicate and reported as the average percent luciferase activity. Purified IgG of monoclonal brNAbs 4E10, 2G12 and IgGb12 were obtained from Polymun Scientific (Austria). PGT121 and PGT126 were obtained from the International AIDS Vaccine Initiative.
Transfection supernatants containing WT or ΔNINN/WL/KH/DE Env pseudotyped luciferase reporter viruses were serially diluted and then used to infect U87.CD4.CCR5 target cells. The cells were lysed at 48-h post-infection and luciferase activity measured.
Sensitivity of ΔNINN/WL/KH/DE Env Pseudotyped Luciferase Reporter Viruses to brNAbs
The ΔNINN/WL/KH/DE mutation conferred sensitivity to various brNAbs whose properties are listed in
The combination of the ΔN139INN gp120 V1 mutation and the WL/KH/DE gp41 mutations resulted in the sensitization of the viral gp120-gp41 complex to neutralization by oligomannose glycan-dependent gp120-directed brNAbs and an MPER-directed brNAb. It is proposed that the ΔN139INN mutation alters the conformation of the glycan shield of gp120, enabling better access for brNAbs to oligomannose-dependent epitopes on the Env surface, while the gp41 mutations within the gp120-gp41 association site and MPER may lead to a more open Env structure, and exposure of brNAb epitopes within the MPER. Alternatively or additionally, WL/KH/DE may be associated with structural change in the MPER itself, with increased MPER flexibility and/or altered membrane interactions facilitating paratope-mediated extraction of the 4E10 epitopes from the envelope. These proposed changes to the MPER may be present in cell-free virions or could occur during the virus-cell fusion process.
The neutralization assays were repeated using additional brNAbs: VRC01, directed to the CD4-binding site of gp120 and 10E8, directed to the MPER of gp41 (1,2). The VRCO1 and 10E8brNAbs are of particular interest because they exhibit greater HIV-1 neutralization potency and neutralization breadth when compared with IgGb12 and 4E10, respectively. The data presented in
These data indicate that the combination mutation, ΔNINN/WL/KH/DE, sensitizes the viral gp120-gp41 complex to neutralization by oligomannose glycan-dependent gp120-directed brNAbs(PGT121 and PGT126) and by an MPER-directed brNAb (10E8).
It was proposed that incorporation of ΔNINN/WL/KH/DE into a human immunodeficiency virus like particle (HIVLP) immunogen enhanced the presentation of oligomannose-dependent epitopes in gp120 as well as MPER epitopes in gp41 in a quasi-native context and in conformations that would promote the production of brNAbs in vaccinated mammals including humans. To test this proposal, a pcDNA3-based GagPol expression vector, pcGagPolVpu was constructed for HIVLP production. The vector contains the HIV-1NL4.3(3) gagpol-vif-vpu genomic fragment. Translation termination codons were introduced to the vif and vpr open reading frames close to the initiation codons in order to block their expression. Furthermore, a gene fragment encoding the Rev-responsive element was PCR-amplified using the pNL4.3 infectious clone as template and ligated into the unique XbaI site in pcGagPolVpu downstream of the HIV-1 coding region. The inclusion of the Rev responsive element is to enable nuclear export of mRNAs encoding Gag, GagPol and Vpu, which is mediated by the viral protein Rev.
HIVLPs were produced by co-transfecting 293T cells with pcGagPolVpu (1 μg) plus pΔKAD8-WT (1 μg), or pcGagPolVpu (1 μg) plus pΔKAD8-ΔNINN/WL/KH/DE (1 μg) using the Fugene HD procedure. Control HIVLPs lacking Env were produced by cotransfecting 293T cells with pcGagPolVpu (1 μg) plus pCMV-Rev (1 μg), a vector that expresses the viral protein Rev from a cytomegalovirus promoter (4). At 4-8 h post-transfection, the culture medium was replaced with Optimem and the supernatants harvested at 72 h post-transfection. HIVLPs were partially purified by ultracentrifugation (25,000 rpm for 2 hr at 4° C. in a SW41 rotor) through a 1.5 ml sucrose cushion (25% sucrose in PBS). The pelleted virions were resuspended in PBS and then subjected to SDS-PAGE under reducing conditions followed by Western blotting with a sheep polyclonal antiserum raised to recombinant gp120 (DV-012) and IgG purified from the plasma of an HIV-1-infected individual (HIV+IgG). Western blotting with DV-012 shows that the HIVLPs contain mature gp120 as well as the uncleaved Env precursor, gp160. Blotting with HIV+IgG reveals the presence of Gag and GagPol cleavage products in addition to gp120 and gp160 (
An enzyme linked immunoassay (ELISA) protocol was developed to determine whether the ΔNINN/WL/KH/DE mutations led to enhanced recognition of pseudovirion-incorporated Env by brNAbs. The volumes of HIVLP suspensions were adjusted with PBS to normalize the capsid (CA) based on the intensity of the CA band observed in the western blot. 96-well ELISA plates (NUNC) were coated with 50 μl HIVLP suspensions at 37° C. for 2 h. The plates were washed thrice with PBS and then blocked with 100 μl of a 3% bovine serum albumin-PBS solution at 37° C. for 1 h. The plates were again washed three times prior to the addition of a dilution series of PGT121 in 50 μl 5% skim milk powder-PBS. The plates were incubated for 1 h at 37° C. The plates were washed 6 times with KPL buffer lacking Tween 20 prior to the addition of 50 μhorseradish peroxidase conjugated rabbit immunoglobulins to human immunoglobulins (DAKO) in 5% skim milk powder-PBS. The plates were incubated at room temperature for 1 h and then washed 6 times with KPL buffer lacking Tween 20. The ELISA was developed with 3,3′,5,5′-tetramethylbenzidine in phosphate-citrate buffer (pH 5.0) and the reaction terminated with 1N HCl. The background absorbance at 620 nm was subtracted from the absorbance at 450 nm. The data (
The overall coating levels of WT, ΔNINN/WL/KH/DE and Env-deficient HIVLPs were compared in a modified ELISA employing the anti-CA mouse monoclonal antibody 183-H12-5C (183)(5). In this modified assay, plate-bound HIVLPs were treated with 1% Triton X100 at 4° C. for 1 h to permeabilize the cholesterol-rich envelope to allow mAb 183 to access the internal CA. The plate was washed three times with PBS and a mAb 183 dilution series (50 μl in 5% skim milk powder-PBS) added to the plates. The plates were incubated for 1 h at 37° C. The plates were washed 6 times with KPL buffer lacking Tween 20 prior to the addition of 50 μl horseradish peroxidase conjugated rabbit immunoglobulins to mouse immunoglobulins in 5% skim milk powder-PBS. The plates were incubated at room temperature for 1 h and then washed 6 times with KPL buffer lacking Tween 20. The ELISA was developed as described above. Comparable binding by mAb 183 to WT and ΔNINN/WL/KH/DE-containing HIVLPs as well as to HIVLPs lacking Env indicates comparable coating levels for the 3 HIVLP preparations. Therefore, the observed increased binding by PGT121 to ΔNINN/WL/KH/DE Env-containing HIVLPs is indeed consistent with the enhanced exposure of brNAb epitopes.
It is proposed that incorporation of ΔNINN/WL/KH/DE into a HIVLP immunogen enhances the presentation of oligomannose-dependent epitopes in gp120 and in conformations that promote the production of brNAbs to such epitopes in vaccinated mammals including humans. It is also proposed that incorporation of ΔNINN/WL/KH/DE in a quasi-native context of HIVLPs will improve the presentation of MPER-dependent epitopes in gp41 and in conformations that promote the production of brNAbs in vaccinated mammals including humans.
The disclosure of every patent, patent application, and publication cited herein is hereby incorporated herein by reference in its entirety.
Many modifications will be apparent to those skilled in the art without departing from the scope of the present invention.
The present application is a U.S. national phase of PCT Application No. PCT/AU2014/000287 filed on 17 Mar. 2014, which claims priority from U.S. Provisional Patent Application No. 61/852,179 filed on 15 Mar. 2013, the disclosure of which is included herein by reference in its entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/AU2014/000287 | 3/17/2014 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 61852179 | Mar 2013 | US |
