Patent Application
20120121633
The invention relates to the fields of vaccination and immunotherapy with particular respect to the human immunodeficiency virus.
Conventional Human Immunodeficiency Virus (HIV) vaccine approaches have proved ineffective because the immunodominant viral epitopes are mutable and the conserved epitopes necessary for infection are not sufficiently immunogenic. The CD4 binding site (CD4BS) of the HIV envelope protein of gp120 is a conserved sequence that is essential for viral entry into host cells. However, the development of an effective vaccine against this conserved epitope has been hindered for the following reasons.
The CD4BS core overlaps the B cell superantigen (SAg) site of gp120 recognized noncovalently by the framework regions (FRs) of antibodies (Abs) produced without prior HIV exposure, which results in super antigen suppression of an anti-CD4BS immune response due to defective IgM→IgG class switching and premature apoptosis rather than the desired B cell response. Study of HIV-infected humans and mice immunized with purified gp120 revealed down-regulated adaptive synthesis of Abs to the 421-433 CD4BS region characterized by deficient IgM→IgG class-switching. Nevertheless, long-term survivors of HIV infection do produce Abs to the 421-433 CD4BS region that neutralize genetically diverse HIV strains. While the development of such antibodies in these patients is very slow, continuing over decades during the course of infection, it shows that their endogenous production is not biologically impossible. The lack of proper conformational mimicry and stability by candidate CD4BS immunogens is believed to be another barrier to vaccines employing this epitope.
What is needed and provided herein are new immunogens and immunogenic compositions based on the core CD4BS sequence of gp120 that are effective in rapidly producing neutralizing Abs against HIV in mammals.
One embodiment of the invention provides a synthetic immunogen with a conformation similar to the conformation of the CD4 binding site of gp120 on the surface of HIV that induces neutralizing antibodies to genetically diverse Group M HIV-1 strains, having the formula
L-E
wherein L is a peptide sequence that comprises fourteen or more amino acids selected from the region of gp120 containing the consensus amino acids 406-459 numbered according to the HXB2 numbering system, or a mimotope thereof, and E is an electrophilic group covalently linked to an amino acid side chain of L, having the formula
Y-Y′-Y″
wherein
Y is an electrophilic group,
Y′ is a charged or neutral group, or is absent, and
Y″ is a linker, covalent bond or atom.
The synthetic immunogen is effective to induce the synthesis of HIV neutralizing antibodies to the 421-433 CD4 binding site sequence by recognition of the framework regions of B cell receptors. In one variation L comprises peptide sequence 416-433 of gp120. In another variation L comprises peptide sequence 414-439.
In a related embodiment of the invention, the synthetic immunogen has the formula:
Cys-Leu-Pro-Ser-Arg-Ile-Lys(X)-Gln-Ile-Ile-Asn-Met-Trp-Gln-Glu-Val-Gly-Lys(X)-Ala, or
Ile-Thr-Cys-Leu-Pro-Ser-Arg-Ile-Lys(X)-Gln-Ile-Ile-Asn-Met-Trp-Gln-Glu-Val-Gly-Lys(X)-Ala-Met-Tyr-Ala-Pro-Pro-Ile,
wherein X is an electrophilic group of formula:
and Lys(X) indicates X is covalently linked to a side chain of the lysine residue.
A further embodiment of the invention provides a synthetic immunogen with a conformation similar to the conformation of the CD4 binding site of gp120 on the surface of HIV that induces neutralizing antibodies to genetically diverse Group M HIV-1 strains, having the formula
L-E
wherein L is a peptide sequence that comprises fourteen or more amino acids selected from the region of gp120 containing amino acids 406-459 with one or more amino acid sequence differences compared to the consensus sequence of amino acids 406-459 of Group M HIV-1 gp120, and E is an electrophilic group covalently linked to an amino acid side chain of L, having the formula
Y-Y′-Y″
wherein
Y is an electrophilic group,
Y′ is a charged or neutral group, or is absent, and
Y″ is a linker, covalent bond or atom.
The invention also provides immunogenic composition that include an immuogen of the invention and an adjuvant.
In another variation of the above embodiments, L is a peptide sequence that comprises fourteen or more amino acids selected from the region of gp120 containing amino acids 406-459 or a mimotope thereof and a second epitope of gp120 or a mimotope thereof.
The synthetic immunogens of the invention may further include one or more cross-links between amino acids that rigidify the conformation.
The immunogens of the invention may be conjugated to one or more carrier molecules. The carrier molecule may, for example, be the carrier molecule is selected from the group consisting of keyhole limpet hemocyanin, tetanus toxoid, and CD40 ligand. The carrier molecule promotes folding of the synthetic immunogen into a conformation similar to the conformation of the CD4 binding site of gp120 on the surface of HIV.
Another embodiment of the invention provides a method for producing HIV neutralizing antibodies to genetically diverse Group M HIV-1 strains within an organism capable of producing antibodies, such as a mammal, for example, a mouse, rabbit, monkey or human including the step of: administering an immunogen of the invention to the organism in an amount effective to cause production of neutralizing antibodies against the CD4-binding site of HIV gp120. The administering step may further include administering an adjuvant to the mammal.
A further embodiment of the invention provides a method for producing HIV neutralizing antibodies to genetically diverse Group M HIV-1 strains within an organism capable of producing antibodies, such as a mammal, for example, a mouse, rabbit, monkey or human including the step of: administering an immunogen according to the invention to the organism in an amount effective to cause production of neutralizing antibodies against the CD4-binding site of HIV gp120. The administering step may further include administering an adjuvant to the organism.
A further embodiment of the invention provides a method for the preparation of an immunogen with a conformation similar to the CD4 binding site of gp120 on the surface of HIV that induces neutralizing antibodies to genetically diverse Group M HIV-1 strains, said immunogen having the formula
L-E
wherein L is gp120 and E is an electrophilic group conjugated to a side chain functional group of L having the formula
Y-Y′-Y″
wherein
Y is an electrophilic group,
Y′ is a charged or neutral group, or is absent, and
Y″ is a linker, covalent bond or atom, comprising the steps of:
(a) conjugating varying numbers of Y-Y′Y″ groups per molecule of gp120;
(b) incubating the resultant L-E preparation for a length of time sufficient to enable intermolecular covalent bonding between the L-E molecules
(c) fractionating the L-E preparation into multiple fractions containing individual subpopulations of L-E molecules characterized by their size, charge, hydrophobicity or conformation;
(d) assaying the several variant L-E fractions from step (c) to determine their CD4 binding activity or their antibody binding activity; and
(e) identifying those variant L-E fractions from step (c) that induce the greatest synthesis of HIV neutralizing antibodies in an organism.
Another embodiment of the invention provides a method for the preparation of an immunogen that induces neutralizing antibodies to genetically diverse Group M HIV-1 strains, said synthetic immunogen composed of intact HIV-1 particles having on their surface molecules with the formula
L-E
wherein L is gp120 and E is an electrophilic group conjugated to a side chain functional group of L having the formula
Y-Y′-Y″
wherein
Y is an electrophilic group,
Y′ is a charged or neutral group, or is absent, and
Y″ is a linker, covalent bond or atom, comprising the steps of:
(a) conjugating varying numbers of Y-Y′Y″ groups per gp120 molecule expressed on the surface of intact HIV-1;
(b) incubating the resultant HIV-1 particles for a length of time sufficient to enable intermolecular covalent bonding between the surface L-E molecules
(c) fractionating the HIV-1 particles with surface L-E molecules into multiple fractions containing individual subpopulations of HIV-1 particles characterized by their size, charge, hydrophobicity or conformation;
(d) assaying the several variant HIV-1 fractions from step (c) to determine their CD4 binding activity or their antibody binding activity; and
(e) identifying those variant HIV-1 fractions from step (c) to induce the greatest synthesis of HIV neutralizing antibodies in an organism.
Another embodiment provides an isolated polypeptide, such as but not limited to an antibody, comprising the framework regions (non-underlined portions) of at least one of the following antibody VL and VH amino acid sequences:
SKLASGVPGRFSGSGSGNSYSLTISSMEAEDVATYYCFQGSGYPYTFGGG
IHPNSGNTNYNEKFKGKATLTVGTSSSTAYVDLSSLTSEDSAVYYCARPG
ASQSISGIPSRFSGSGSGTDFTLSINSVETEDFGMYFCQQSNSWPLTFGA
VIWSGGSTDYNAAFISRLSISKDNSKSQVFFKMNSLQANDTAIYYCARTG
FAYWGRGTLVTVS;
STSNLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCHQYHRSPRTFG
IRNKANGYTTEYSASVKGRFTISRDNSQSILYLQMNTLRAEDSATYYCAR
DNQSFYYAMDYWGQGTSVTVSS;
TFGGGTKLEIK;
IWRGGSTDYNAAFMSRLSITKDNSKSQVFFKMNSLQADDTAIYYCAKRYG
NYGGGAMDYWGQGTSVTVSS;
SNLASGVPARFSGSGSGTSYSLTISRMEAEDAATYYCQQRSSYPYTFGGG
IRSKSNNYATYYADSVKDRFTISRDDSQSMLYLQMNNLKTEDTAMYYCVR
ERAGYFDVWGAGTTVTVSS;
GSTLQSGIPSRFSGSGSGTDFTLTISSLEPEDFAMYYCQQHNEYPYTFGG
ISCYNGATSYNQKFKGKATFTVDTSSSTAYMQFNSLTSEDSAVYYCARGG
TTVVATGKYAMDYWGQGTSVTVSS;
IWSGGSTDYNAAFISRLSISKDNSKSQVFFKMNSLQANDTAIYYCARNKD
YGSSYDYYAMDYWGQGTSVTVSS;
WTFGGGTKLEIK;
IYPGSGGTAYNQKFKGKATLTADKSSSTAYMELSSLTSEDSAVYYCTKFR
FSSFAMDYWGQGTSVTVSS;
WTFGGGTKLEIK;
FHPGSGGGAYSQKFKGKATLIADKSSSIAYMEVISLTSEDSAVYYCTRFR
YSSFAMVYWGQGTSVTVSS,
wherein underlined sequences are CDRs and non-underlined sequences are antibody framework regions. A related embodiment provides an isolated polypeptide such as but not limited to an antibody that includes at least one of the aforementioned VH or VL sequences. The aforementioned polypeptides may be administered to a human for the prophylaxis or treatment of HIV infection.
Another embodiment of the invention provides an isolated preparation of polyclonal antibodies obtained following immunization of an organism capable of producing antibodies, such as a mammal, with an immunogen or immunogenic composition.
A further embodiment of the invention provides a method of prophylaxis or treatment against HIV infection that includes the step of administering an immunogen or immunogenic composition as described herein to human being in an amount effect to induce the production of HIV neutralizing antibodies. Use of an immunogen or immunogenic composition as described herein for the prophylaxis or treatment of HIV infection in a human is similarly provided.
Another embodiment of the invention provides a method of prophylaxis or treatment of HIV infection that includes the step of administering to a human monoclonal or polyclonal antibodies as described herein or polypeptides. Use of such antibodies for the prophylaxis or treatment of HIV infection in a human is similarly provides.
The invention also provides the use of any of the immunogens, antibody sequences, and antibodies described herein for the preparation of a medicament for the prophylaxis or treatment of HIV infection in a human.
Additional features, advantages, and embodiments of the invention may be set forth or apparent from consideration of the following detailed description, drawings, and claims. Moreover, it is to be understood that both the foregoing summary of the invention and the following detailed description are exemplary and intended to provide further explanation without limiting the scope of the invention as claimed.
Table 1. 416-433 epitope recognition by neutralizing IgA from HIV infected patients and a neutralizing murine MAb raised by KLH-E-416-433 immunization. The table shows that the gp120 amino acids important for recognition of CD4 are also critical for recognition of the neutralizing Abs, supporting recognition of the 421-433 CD4BS sequence as the mechanism of neutralization. Sequence conservation determined from 1699 group M strains (http://www.hiv.lanl.gov/). Conservative mutations defined as in BLAST; I/L-P-C-R/K-I-K/R-Q-I/V-I/V-N/D-M/L-W-Q-E/D/Q-V/I-G-K/Q/R-A). Dotted boxes indicate residues important for sCD4 binding (dots) or Ab binding determined by crystallography or by mutagenesis (reduction of binding by >50%). Values for I19-21 IgA and MAb IgM 2G9 correspond to the ratio (IC50wild type peptide/IC50mutant peptide) where IC50 is the concentration of soluble wildtype 416-433 peptide or its Ala mutant at each position displaying 50% competitive inhibition of Ab binding to immobilized KLH-E-416-433. Yellow boxes, non-contacting residues or <50% loss of binding activity upon mutagenesis. sCD4 data are from refs 10 and 11 and PDB structures 1GC1, 1G9M, 1G9N, 2QAD, 2B4C. MAb IgM 2G9 obtained by immunization with KLH-E-416-433.
Table 2. Neutralization of genetically diverse HIV strains by polyclonal serum Abs (unfractionated serum) from a rabbit and mice immunized with KLH-E-416-433. Titer, dilution at which 50% neutralization occurred. Data are from plots of varying serum dilution versus neutralization fitted to the equation % neutralization=100/(1+10(logIC50−log [Ab])·Hillslope). PBMC host cells. The gp120 V domains of these strains are highly divergent (values shown are computed using strain 97ZA009 as reference). Sequence alignments were with ClustalW2 program. NA, not available; Underlined red, non-conservative replacements compared to the immunogen. Underlined green, conservative replacements; X, unidentified amino acid.
Table 3. Neutralization of murine anti-E-416-433 MAbs raised from KLH-E-416-433 immunization. PBMC host cells. Data are from plots of varying MAb concentration versus neutralization. IC50 is defined as the MAb concentration showing 50% neutralization. NA, not analyzed.
Table 4. Neutralization of HIV by serum from a monkey immunized with KLH-E-416-433 followed by E-gp120 (day 176 serum, immunization as shown in
Table 5. Exemplary carrier and adjuvant combinations.
E-421-433. A, Soluble CD4 reactivity of the immobilized electrophilic 421-433 CD4BS sequence mimetics, KLH-E-416-433, KLH-E-414-439 and KLH-E-421-433 (see
Table 6. Fortuitously improved recognition of Ala mutants of the 421-433 CD4BS sequence at P417, Q422, E429 by pooled IgA patients with HIV infection for 19-21 years (I19-21) and murine MAb 2G9 raised by immunization with KLH-E-416-433. Values are ratios of wildtype peptide binding/mutant peptide binding computed as in Table 1.
Production of antigen-specific antibodies (Abs) by adaptively differentiated B lymphocytes is central to protection against microbial infections. The HIV surface is studded with noncovalently associated trimeric gp120-gp41 complexes that have been the targets of numerous vaccine trials with no or little evidence for vaccine efficacy. The problem is the absence of structurally conserved coat protein epitopes essential for the viral life cycle that can induce a sufficient adaptive Ab response. Most anti-HIV Abs bind the mutable, gp120 variable (V) domain epitopes. The virus expresses very few neutralizing epitopes suitable for vaccine targeting.15 The binding site for CD4 on gp120 (CD4BS) is a conformational determinant10,16 containing discrete regions with mostly conserved structure across Group M HIV strains. HIV initiates infection by binding its primary host cell receptor CD4. The CD4BS is vulnerable to host immunity, and rare monoclonal anti-CD4BS Abs with broad neutralizing activity have been cloned from HIV-infected patients.15,17 However, the CD4BS is poorly immunogenic, and the serum of HIV infected patients contains little or no neutralizing activity attributable to anti-CD4BS Abs.
Abs similar to these MAbs are not found at appreciable levels in polyclonal Ab preparations. An HIV vaccine can be effective only if it induces robust polyclonal Abs in biological fluids with the ability to neutralize genetically diverse HIV strains. Several competing hypotheses have been proposed to explain the absence of neutralizing anti-CD4BS Abs in various vaccine strategies. Classical adaptive antibody responses involve very weak antigen binding germline B cell receptors (BCRs; Abs expressed on the cell surface), followed by antigen driven selection of mutant Ab complementarity determining regions (CDRs) that bind antigen with improved affinity. The deficient neutralizing Ab response is suggested to derive from an unusually low affinity of the vulnerable gp120 epitopes for the germline BCRs.18 Conversely, the extreme density of CDR mutations in a rare anti-gp120 neutralizing Ab prompted the suggestion that B cells are intrinsically unable to mount a sufficiently mutated Ab response.19 The CD4BS is a conformationally flexible determinant thought to undergo structural transitions upon dissociation of trimeric gp120 into its monomeric form.20 Individual epitopes within the CD4BS are not equally suited for Ab targeting. Abs that bind the CD4BS of monomeric gp120 but do not neutralize HIV have been described.21,22 The monoclonal Ab b12 binds an epitope overlapping the CD4BS segment located in the gp120 outer domain and expresses comparatively broad neutralizing activity directed against genetically diverse HIV strains.22 An immunogen reverse engineered to present conformational complementarity with the antigen binding site of b12 did not induce neutralizing Abs.23 Similarly, genetically engineered oligomeric gp120 did not induce broadly neutralizing Abs.24
Our approach to HIV neutralization is based on targeting the core of the CD4BS, the region composed of residues 421-433 that provides critical contacts necessary for high affinity CD4 recognition.2,9,10,16 An ample area of 421-433 CD4BS sequence is exposed for interactions with Abs on the gp120 surface according to crystallographyl6,25-30 (
Only limited information is available about the functional importance of the constitutive Abs. As the 421-433 CD4BS sequence component of the gp120-SAg is essential for HIV infection, the Abs may protect against HIV infection if present in sufficient amounts. Indeed, elevated blood levels of 120-SAg binding Abs in non-infected humans are correlated with reduced risk for contracting HIV infection.38 However, sera and purified IgG preparations from the blood of non-infected humans do not neutralize HIV in tissue culture appreciably,39 raising doubt whether the constitutive Abs detected based on binding of purified gp120 and synthetic peptides as antigens can recognize the 421-433 CD4BS sequence on the viral surface with sufficient strength. We previously reported modest HIV neutralization by polyclonal salivary IgA preparations attributed to the 421-433 CD4BS sequence-specific subset of Abs that catalyzed the cleavage of gp120.39 Superior HIV neutralization by catalytic IgAs is consistent with the expectation of increased potency by reuse of a single catalyst molecule for inactivating multiple gp120 molecules. Importantly, polyclonal Abs from blood and saliva examined in these studies are mixtures of diverse Abs, and the strength of HIV neutralization by the polyclonal mixtures will underestimate the neutralizing activity of individual Abs to the 421-433 CD4BS sequence represented at low levels in the mixtures.
If the individual Abs to the 421-433 CD4BS sequence produced by non-infected humans can neutralize HIV with sufficient potency and breadth, tapping the innate Ab repertoire is a potential route to HIV vaccination. The feasibility of this approach depends on overcoming physiological restrictions on amplifying the constitutive Ab subset to a superantigenic epitope. Superantigen binding at the FRs of B cell receptors (BCRs; Abs expressed on the cell surface) generally fails to drive the adaptive differentiation of B cells into plasma cells producing class-switched Abs.4° This is exemplified by the rare production of Abs to the 421-433 CD4BS sequence by animals immunized with gp12041 and HIV-infected humans.42 However, the restriction on producing the Abs is leaky, and certain circumstances supporting an amplified constitutive Ab production have been identified. First, sera from patients with systemic lupus erythematosus contain increased binding Abs to the 421-433 CD4BS sequence,43 and a binding Ab fragment with this specificity and HIV neutralizing activity was isolated from the lupus repertoire.44,45 Second, we reported IgA specific for the 421-433 CD4BS sequence class Abs with potent and broad HIV neutralizing activity that appeared in blood after prolonged HIV infection over two decades.2 Third, a strategy involving covalent binding of electrophilic immunogens to the naturally-occurring nucleophilic sites of BCRs has enabled the high frequency induction of broadly neutralizing murine monoclonal Abs with specificity for the 421-433 CD4BS sequence.12 An overriding advantage of the approach is the potentially correct specificity of the constitutive Ab FRs for the 421-433 CD4BS sequence. Even immunogens that approximate the native 421-433 CD4BS conformation imperfectly can potentially induce amplification of the CD4BS− specific Ab FRs by the induced-fit mechanism (
The present invention overcomes the prior limitations of the art, by employing covalent immunization techniques. The invention provides improved synthetic electrophilic immunogens for HIV vaccination based on the conserved sequence of the 421-433 CD4BS sequence, amino acid nos. 421-433, of HIV gp120 that are effective in inducing the production of HIV neutralizing Abs in mammals, including humans. The probes, control reagents and improved immunogens are shown in
The invention discloses the discovery of deficient IgM→IgG class-switching as the cause of the absent neutralizing Ab response to previously tested vaccine candidate and rectification of this deficiency by immunizations with analogs of gp120, intact HIV and the 416-433 synthetic peptide containing electrophilic phosphonate groups (respectively, E-gp120, E-HIV and E-416-433). The electrophile binds covalently to the nucleophilic sites of secreted preimmune Abs5 and BCRs.46 Each of U.S. Pat. Nos. 6,235,714; 6,855,528; 6,855,804; and 7,524,663 is incorporated by reference herein. These immunogens supported production of class-switched Abs to the 421-433 CD4BS sequence. Neutralizing MAbs raised by immunization with E-gp120 displayed enhanced nucleophilic activity6,13 and binary recognition of the 421-433 CD4BS epitope at the FRs and a second epitope at the CDRS.12 This suggested that the down-regulatory effect of noncovalent 421-433 epitope binding at the FRs can be overcome by: (a) The highly energetic covalent binding of the immunogens to B cells; and (b) simultaneous stimulatory binding of a second epitope at the CDRs. The findings indicated covalent immunization as a viable strategy for inducing broadly neutralizing anti-CD4BS Abs.
Various immunogens with further improvements of conformation are disclosed. Their development entailed maneuvers that rigidify the 421-433 CD4BS peptide sequence alter its three-dimensional folding, including insertion of the electrophilic group, lengthening the sequence by inserting additional amino acids on its flanks, introducing mutations in the sequence and attaching the sequence to a carrier protein molecule that facilitates its folding into a native conformation.
The invention also discloses the discovery of improved conformation of the 421-433 CD4BS expressed by E-gp120 compared to gp120 (
Intact E-HIV expresses the minimally perturbed, native conformational state of the 421-433 CD4BS sequence. The invention discloses subsets of E-HIV variants expressing improved conformational states of the 421-433CD4BS sequence and other epitopes, and the use of the improved E-HIV variants for inducing a neutralizing Ab response to the 421-433 CD4BS sequence.
The invention discloses focusing of the Ab response at the CD4BS using an example electrophilic peptide immunogen that itself binds CD4 (E-416-433).2,12 Immunization of mice and rabbits with E-416-433 conjugated to a carrier protein induced polyclonal Abs that neutralized genetically diverse HIV strains. The carrier protein is an important factor in the invention, as it provides a microenvironment permitting proper folding of the E-416-433 peptide. Consistent with the electrophilic character of the immunogen, the Abs displayed robust catalytic activity.
In macaques, a common primate model for the human immune system, immunization with E-416-413 induced low level neutralizing Abs, sequential immunization with E-416-433 and E-120 induced higher level Abs, and immunization with E-120 also induced the neutralizing Abs. To the inventors' knowledge, the electrophilic immunogens are the first vaccine candidate that induces polyclonal neutralizing Ab responses to a conserved HIV site.
Covalent Vaccination.
Our invention relies on the covalent binding of immunogens containing electrophilic phosphonate groups to nucleophilic Ab sites as the basis for potentially effective HIV vaccination.12 Such nucleophilic sites were originally identified in enzymes of the serine protease family as triads of Ser(Thr)-His-Asp(Glu) residues in which the activated nucleophilic residue (Ser/Thr) forms a covalent intermediate with weakly electrophilic carbonyl group of the substrate. Abs recapitulate this catalytic mechanism, an example of convergent molecular evolution.39,47-49 The strongly electrophilic phosphonate group was incorporated into polypeptide immunogens in the present invention. This enables covalent immunogen binding to nucleophilic BCRs coordinated with specific noncovalent binding of the peptide epitope.5,46 The electrophilic immunogens are designed based on the split-site model, which states that distinct Ab subsites are responsible for initial noncovalent binding and the ensuing nucleophilic attack on the electrophilic carbonyl group of gp120 (FIG. 6).5-8
The reversible binding and catalytic properties of preimmune Abs to the 421-433 CD4BS sequence may provide some level of innate protection against transmission of HIV infection.38,39 However, any such protection comes at a heavy cost. Stimulatory antigens binding at the Ab CDRs drives B cell clonal selection. By contrast, SAg binding at the FRs is thought to downregulate B cell differentiation.50 An impaired adaptive B cell response to the 421-433 epitope is evident from the rare production of Abs that bind peptides spanning this region in HIV infected patients42 and mice immunized with purified gp120 (0.005% of 140,000 MAb clones tested).41 We discovered that an impaired IgM→IgG/IgA class switching is the central defect in the adaptive immune response to the 421-433 CD4BS sequence.
Central points in the covalent vaccination approach are:
a. Covalent Binding. The highly energetic covalent reaction is hypothesized to induce favorable B cell differentiation instead of B cell downregulation due to noncovalent SAg binding (
b. Vaccine Conformation. To induce neutralizing Abs, the immunogen must mimic the native epitope conformation expressed on the HIV surface. One vaccine prototype of the invention, the electrophilic analog of resides 416-433 (E-416-433), binds specifically to CD4, verifying that the 421-433 region is expressed in a native CD4-binding conformation. E-416-433 is also recognized specifically by broadly neutralizing Abs from survivors of HIV infection. Most importantly, the present invention discloses induction of the synthesis of HIV neutralizing polyclonal and monoclonal Abs in animals by E-416-433 conjugated to the carrier protein keyhole limpet hemocyanin (KLH). Although not appreciated widely, the carrier protein influences the conformation of conjugated peptides profoundly. The pitfall of small peptide immunogens is their ability to assume alternate conformations in varying microenvironments. This is exemplified by our report that Abs raised by immunization with the 421-436 peptide conjugated to different polypeptides displayed differing binding specificity.51 If the peptide folds into a non-native conformation, the immune system will not amplify or improve the innate epitope recognition capability of the Ab FRs. The carrier protein is important because it provides the local microenvironment composed of neighboring amino acids that induce flexible peptides into distinct conformations. E-416-433 also contains the LPSR1 residues at the N terminus, which stabilizes the conformation of the 421-433 region.52,53 The side chain chemical modification of the peptide with phosphonate groups is also a significant feature as it improves binding to CD4 and Abs by imparting rigidity to the epitope mimetic. Since E-416-433 binds CD4 ˜100-fold more strongly than previously tested 421-433 region peptides, it is predicted to induce a superior neutralizing Ab response. Mutagenesis studies showed that key amino acids necessary for the CD4 binding function of E-416-433 are also essential recognition elements of the Ab epitope. Previous studies on Abs to peptide immunogens containing part or all of the 421-433 region but without the essential features of KLH conjugated E-416-433 conjugated KLH failed to induce sufficiently neutralizing Abs to the 421-433 CD4BS region.42,54-56 Importantly, as the previously tested vaccine candidates did not mimic the native 421-433 CD4BS conformation sufficiently, they induced the classical CDR-based response. The CDR-based response results in production of non-neutralizing Abs because of induced-fit considerations shown in
In addition to the carrier protein conjugated peptides, the invention discloses improved variants of full-length E-gp120 and E-HIV that offer the advantage of improved 421-433 CD4BS sequence conformation suitable to amplify the FR-based recognition site of Abs. Yet another advantage of the improved E-gp120 and E-HIV variants is the availability of additional epitopes for binding the CDRs. A second epitope that binds the CDRs simultaneously with binding of the 421-433 CD4BS sequence to the FR-based sites is helpful, because such a binary binding reaction overcomes the negative cellular signaling associated with binding of the FRs alone (
c. Catalytic Activity. Covalent immunization offers the bonus of strengthened Ab nucleophilic reactivity by virtue of adaptive selection of BCRs that bind the electrophile most strongly.6,13,57 In turn, the enhanced nucleophilicity improves HIV inactivation as follows (
Preclinical Ab Protection Assays.
Tissue culture infection models are indispensable to assess the potency and breadth of neutralizing activity of Abs induced by candidate vaccines.59 CCR5-dependent primary strains are substantially more resistant to most Abs directed to the HIV proteins compared to lab-adapted strains.60 Abs to the 421-433 CD4BS epitope neutralize the most ‘difficult-to neutralize strains’ in the classical peripheral blood mononuclear cell (PBMC)/clinical isolate infection assay. This assay is the closest available tissue culture model for the natural infection process. Engineered reporter cell lines and pseudovirions have been developed for convenient analysis of large numbers of Ab samples, e.g., the TZM-B1 cell line/pseudovirion assay.61 Several publications have noted discrepant neutralizing activities of anti-HIV MAbs in the PBMC/clinical isolate and TZM-B1/pseudovirion assays.22,62,63 Excessive expression of the coreceptor CCR5 on TZM-B1 cells compared to PBMCs is cited as a potential reason for discrepancies (˜1000-fold difference of CCR5 levels).64 The conformational flexibility of gp12020,26,65 in differing membrane microenvironments is another variable. Variations in the conformations of the epitopes expressed by clinical HIV isolates versus pseudovirions are conceivable.
HIV infects chimpanzees transiently. The infection does not progress to AIDS. Immunization of chimpanzees with recombinant gp120 suppressed HIV viremia, but human trials of the gp120 immunogen did not reduce HIV infection risk.66-68 As the HIV and SIV envelope proteins are structurally divergent, direct testing of candidate HIV vaccines in the SW-infection model is difficult. Hybrid simian-human virus strains (SHIN) containing the HIV envelope proteins grafted into SW produce viremia in rhesus monkeys. Candidate vaccines that induced cytotoxic T cells protected monkeys from SHIV infection but did not protect humans from HIV infection.69 The SHIV/rhesus monkey model was recently suggested to be a useful ‘gatekeeper’ to identify candidate vaccines that induce ‘better immunity’ compared to the failed immunogens.70 One use of the SHIV-monkey model is to determine the circulating/mucosal titers of Abs needed to prevent or reduce the acute stage of infection in vivo. Moreover, the immune system of monkeys may be a good model of the human response to candidate vaccines compared to phylogenetically lower species. Therefore, monkey studies were done to validate the present invention.
The present invention developed and utilized the following electrophilic immunogens: E-120, intact E-HIV and synthetic E-peptides containing the 421-433 peptide region. Improved E-peptide and E-gp120 variants have been identified as the prototype vaccine candidates based on the properties of Abs induced by these immunogen in mice, rabbits and monkeys. The studies also resulted in unexpected findings of: (a) down-regulated adaptive immunity attributable to the SAg character of the 421-433 CD4BS epitope; and (b) upregulation of the adaptive immune response by covalent stimulation of B cells with the electrophilic immunogens.
The following findings were made, as discussed in further detail below:
Preimmune IgM and IgA from humans/experimental animals express the innate capability to recognize the 421-433 region by noncovalent means and proceed to catalyze the cleavage of gp120.
Adaptive synthesis of Abs to the 421-433 is down-regulated upon HIV infection/immunization with gp120.
Survivors of prolonged HIV infection mount a slow Ab response to the 421-433 epitope with eventual production of Abs that neutralize genetically diverse HIV strains with exceptional potency.
Covalent immunization with improved electrophilic immunogens overcomes the B cell down-regulation. The prototype vaccine E-416-433 conjugated to KLH induces the production of a focused anti-CD4BS Ab response that neutralizes diverse HIV strains.
E-gp120 and its improved variants induce Abs that neutralize diverse HIV strains.
Immunization with E-416-433 and E-120 results in improved covalent binding and catalytic hydrolysis of gp120 by Abs.
Use of E-416-433 for Improved Detection of Preimmune ABS to 421-433 CD4BS Epitope.
The peptide component of E-peptides bind reversibly to the traditional noncovalent binding site of Abs. In addition, the electrophilic phosphonate component binds the nucleophilic sites of catalytic Abs irreversibly. We reported previously the cleavage of gp120 by catalytic IgM and IgA class Abs from humans without HIV infection (preimmune Abs) that was selectively inhibited by the short E-421-433 peptide, indicating the importance of the 421-433 region for initial noncovalent binding.9,39 Preimmune SIgA from saliva neutralized HIV with modest potency. The rank orders of neutralization potency and gp120 cleavage rates were the same: SIgA>serum IgA>>serum IgG.39 This is consistent with more efficient virus inactivation by catalysts compared to reversibly-binding Abs.
We used the longer E-416-433 peptide to isolate highly neutralizing Abs from preimmune human and murine Abs also Enriched HIV neutralization of the epitope-specific Abs isolated from human and mouse serum by affinity chromatography on agarose-conjugated E-416-433 was evident (
Trace protease contamination in various catalytic Ab preparations tested by our group was ruled out as follows: Fab fragments retained the activity;9 the activity was not removed by denaturing gel filtration, a procedure that frees Abs of noncovalently-associated proteins;9,39 the Abs were purified to constant catalytic activity by sequential chromatography steps;9 active site titration indicated that the number of catalytic sites corresponds to the predicted number of sites;39 unlike conventional proteases, the Abs hydrolyzed gp120 specifically;6,9,39 recombinant Abs with 421-433 CD4BS sequence binding activity hydrolyzed gp120;37,45 and catalytic monoclonal Abs were obtained by immunization with electrophilic gp120.6 We have also mapped the catalytic site of Abs by mutagenesis47 and VL-VH domain shuffling.72,73 The catalytic site of one of our Abs was identified by crystallography.48
Lupus patients mount Ab responses that are normally disfavored in humans with autoimmune disease. HIV infection occurs rarely in lupus patients. HIV neutralizing Ab fragments specific for the 421-433 region prepared from lupus patients without HIV infection were reported.37,44,45 These fragments recognize E-416-433 strongly, e.g., the VH3 family single chain Fv (scFv) JL427 binds KLH-E-416-433 with Kd 16 nM. VH3 family Abs are thought to recognize the gp120 SAg site preferentially.34 We replaced the FR1, FR3 and CDR1 of the VH4-family scFv clone GL2 with the corresponding VH3-family scFv JL427 regions (
Use of E-416-433 for Discovery of Impaired Class Switching and Slow, Infection-Induced Adaptive AB Response.
The mature systemic Ab response is normally dominated by IgG Abs. In contrast, IgMs dominated the Ab response to BSA-E-416-433 in HIV infected patients despite prolonged infection (0.5-5 years; n=10;
That IgA fractions from humans without HIV infection display superior HIV neutralization compared to the IgG fractions has been reported.39 Therefore, the neutralization of a heterologous subtype C primary HIV isolate by purified IgA from the blood of 12 patients infected for varying durations with presumptive subtype B HIV strains (0.5-21 years, all patients from the USA; subtype B infection was confirmed for 3 patients infected for 19-21 years by sequencing the gp120 gene2) was tested. The subtype C HIV strain was selected to minimize detection of Abs to the gp120 V domains, as such Abs do not neutralize genetically heterologous HIV strains. The subtype C strain is not neutralized by murine Abs to subtype B full-length gp120. The sequences of its V domains diverge substantially from the autologous subtype B strains identified in the I19-21 patients (e.g., the V3 domain epitope shown in
The slow but distinct Ab response to the 421-433 CD4BS response can be interpreted favorably with respect to the prospect of therapeutic vaccination, as the response will generate CD4BS-specific memory B cells. The present invention provides immunogens that rapidly induce HIV neutralizing Abs against the CD4BS. The immunogens can be used to amplify the CD4BS-memory B cells found in HIV infected patients as a therapeutic vaccine strategy.
The functional properties of IgA from 3 survivors of prolonged infection (19-21 years; I19-21 patients) has been reported.2 Affinity chromatography of IgA from these patients on immobilized E-416-433 yielded epitope-specific eluates with enriched E-416-433 binding activity. The subtype C strain was neutralized by the noncovalently bound IgA obtained by acid elution and covalently bound IgA obtained by PAM elution more potently than the starting IgA (respectively, by 151-fold and 4688-fold
The covalent binding of electrophilic phosphonates placed into our E-immunogens to B cell receptors and secreted Abs was reported previously.5,6,46 In the present invention, immunization with E-gp120 induced a robust IgG response to the 421-433 CD4BS epitope detected using BSA-E-416-433 as the antigen in ELISA tests (
Rectification of the deficient class switching is consistent with our report of high frequency induction of monoclonal IgGs by immunization with E-gp120.12 Seven of 17 anti-E-gp120 MAbs displayed neutralizing activity attributable to 421-433 CD4BS epitope recognition. The MAbs neutralized genetically divergent clinical HIV isolates (n=11 strains), including all ‘difficult-to-neutralize’ strains tested (2 tier 1 strains and 3 tier 2 strains). Of the 7 neutralizing MAbs, 6 displayed binary-epitope reactivity. That is, the same MAb was able to bind two peptide epitopes that are spatially separated in the gp120 crystal structure (residues 301-311 and 421-431). Monovalent Fab and scFv fragments also displayed the binary epitope reactivity. The crystal structure of a Fab fragment indicated an antigen binding cavity formed by the CDRs flanked by another cavity formed by VH FR residues previously implicated in gp120 binding by preimmune Abs (FIG. 15A).35,36 Site-directed mutagenesis at a residue in the FR-cavity reduced the binding of E-416-433, confirming recognition of the 421-433 CD4BS epitope at this cavity (
As noted previously, Abs to the 421-433 CD4BS sequence are produced very rarely by immunization with monomeric gp120 devoid of electrophilic groups.41 E-gp120, however, present a strongly immunogenic 421-433 CD4BS epitope on its surface. Immunoadsorption of the E-gp120 immunogen-induced polyclonal Abs on the E-416-433 probe removed 66% of E-gp120 binding Abs (
E-gp120 Variants
E-gp120 is not a homogeneous immunogen. It is prepared by insertion of electrophilic phosphonate groups into the side chains of surface Lys groups of gp120. It is commonly assumed that the starting monomer gp120 molecules into which the electrophiles are inserted represent a single, conformationally homogeneous population. Given the conformational heterogeneity of the CD4BS and other segments of gp120,26,52 this assumption may be incorrect, in which case, the conformationally distinct starting monomer gp120 molecules will give rise to conformationally distinct E-gp120 monomers. Heterogeneity is also created by the electrophile insertion step. On average, about 70% of surface accessible Lys side chains are linked chemically to the electrophile. The number of electrophiles per molecule of gp120 may be assumed to be distributed in a Gaussian fashion, with subsets of molecules containing differing numbers of the electrophiles. Moreover, insertion of the electrophile into gp120 results in intermolecular covalent bonding between the monomeric gp120 molecules, resulting in formation of various oligomeric species, including well-defined trimers and dimers (
Each of the foregoing mechanisms holds potential for altering the CD4BS conformation in subtle but important ways.
The present invention discloses the use of improved E-gp120 variant species (IE-gp120) for inducing neutralizing Abs to the CD4BS. Methods for isolating and using the IE-gp120 variant species expressing the CD4BS in a conformation resembling the native viral CD4BS are also disclosed. Improved mimicry of the native CD4BS by the immunogens can be anticipated to induce improved neutralizing Abs.
As E-120 oligomers are stably linked, fractionating various IE-gp120 species is routine (
The IE-gp120 species may be tested for immunogenicity in experimental animals as in Example 2, Example 4-8 and Examples of Methods. The immunogenicity tests include measurement of the ability of induced Abs to bind the 421-433 CD4BS sequence, catalyze the hydrolysis of gp120 and neutralize genetically diverse HIV strains. In addition, the Abs may be capable of removing virus through certain Fc-dependent functions, for example, Ab-dependent cellular virus inhibition.75 Therefore, the sera may also be tested for Fc-dependent viral removal using appropriate assays to determine the mechanism of Ab action.
E-HIV and E-HIV Variants
Like the E-gp120 immunnogen, the intact E-HIV immunogen described in Example 2 overcomes the problem of deficient Ab class switching and induces a class-switched Ab response directed at the 421-433 CD4BS sequence.
Another advantage of the E-HIV immunogen is the that it expresses gp120 trimers with the 421-433 CD4BS sequence expressed in its native conformation with minimal perturbation due to insertion of the electrophilic groups. Thus, E-HIV is likely to induce Abs that recognize the neutralization-relevant conformation of the 421-433 CD4BS sequence.
Once electrophilic phosphonates are incorporated into the amino acids side chains of the virally expressed gp120, intermolecular covalent bonding of the gp120 molecules composing the trimeric gp120 complexes may occur by the same mechanisms as in the case of E-gp120 covalent self-assembly, that is, by means of the covalent reaction between the electrophilic phosphonate and a naturally occurring nucleophilic amino acid of gp120.
As in the case of side chain labeling of purified gp120, varying numbers of the electrophilic group are incorporated into the side chains of virally expressed gp120, creating heterogeneity with respect to conformation. Similarly, heterogeneity with respect to the degree of covalent oligomerization of the trimeric gp120 on the viral surface is also likely.
In addition to gp120, the electrophilic groups will also be incorporated into the side chains of other proteins expressed on the viral surface.
The present invention discloses the use of E-HIV and its improved variant species (IE-HIV) for inducing neutralizing Abs to the CD4BS. Methods for isolating and using the E-HIV and IE-HIV variant species expressing the CD4BS in a conformation resembling the native viral CD4BS are also disclosed. Improved mimicry of the native CD4BS by the immunogens can be anticipated to induce improved neutralizing Abs.
E-HIV is prepared using inactivated HIV from a suitable virus strain, for example strain MN. The HIV-1 particles in the supernatants of cell culture supernatants are purified by precipitation using 1-2% polyethylene glycol (PEG) or another method such as gel filtration chromatography. This removes soluble proteins, including monomer gp120 shed from the virus. Inactivation is done using psoralen and UV light or 2-aldrithiol, methods that minimize disruption of the native surface structure of HIV-1. Insertion of electrophilic phosphonate groups into Lys side chains of surface proteins expressed on the surface of HIV particles is done essentially as described for purified E-gp120 preparation using a neutral pH buffers. The E-HIV is purified by PEG precipitation, the extent of phosphonate insertion per unit protein mass of the E-HIV is determined, and the E-HIV is tested as an immunogen in experimental animals as described in Example 1 and 3.
To obtain improved E-HIV (IE-HIV) variants expressing the minimally perturbed, native 421-433 CD4BS conformation, the E-HIV is fractionated as described for E-gp120. One embodiment of the invention provides one or more fractions of IE-HIV, such as one or more column fractions. Fractionation may be performed by one or more of various routine methods, for example, by resolutive size, charge and/or hydrophobic HPLC methods (e.g., Superose, Mono Q and hydroxylapatite columns). In addition, affinity chromatography using immobilized neutralizing Abs or immobilized CD4 may be employed to identify the IE-HIV species expressing the most favorable CD4BS conformation. For example, the IE-HIV species may be isolated by binding to the highly-neutralizing scFv JL427 or IgA from patients with very prolonged HIV infection. As these Abs recognize the native conformation of the 421-433 CD4BS sequence, the procedure identifies the IE-HIV species expressing the native 421-433 CD4BS conformation.
The IE-HIV species may be tested for immunogenicity in experimental animals as in Example 2, Examples 4-8 and Examples of Methods. The immunogenicity tests include measurement of the ability of induced Abs to bind the 421-433 CD4BS sequence, catalyze the hydrolysis of gp120 and neutralize genetically diverse HIV strains. In addition, the Abs may be capable of removing virus through certain Fc-dependent functions, for example, antibody-dependent cellular virus inhibition.75 Therefore, the sera may also be tested for Fc-dependent viral removal using appropriate assays to determine the mechanism of antibody action.
In view of its behavior as an improved probe for neutralizing Abs to the 421-433 CD4BS epitope described in Example 1, the single epitope E-416-433 provided the opportunity to induce an Ab response focused on the 421-433 CD4BS sequence. This may potentially be a useful feature, as certain other epitopes of gp120 can induce undesirable Abs that can enhance infection.
The present invention discloses that covalent immunization with the peptide KLH-conjugated Cys-E-416-433 alone is sufficient to induce neutralizing Ab synthesis. As E-416-433 is a small and flexible peptide, the constraints placed on its conformation by the carrier protein microenvironment are critical. Although most investigators consider the carrier to be a routine component of vaccines, this does not apply to the E-416-433 immunogen. Different carriers may constrain the peptide into different conformations.51 Consequently, we took care to attach E-416-433 to KLH only via its terminus. The peptide immunogen was prepared by conjugating the N terminal Cys residue with Lys residues of KLH using the bifunctional reagent 4-maleimidobutyric acid N-hydroxysuccinimide ester. The resultant KLH-E-416-433 conjugate contained 2000 copies of E-416-433/molecule of KLH. The density of the peptides is also important for inducing an Ab response. Very low density will only induce weak, monovalent cellular signaling through the B cell receptor, whereas excessive density may increase signalizing that is too strong and results in B cell tolerance.
BALB/cJ mice and New Zealand White Rabbits were immunized with KLH-conjugated Cys-E-416-433 by the intranasal route and subcutaneous routes, respectively. Specific primary and secondary polyclonal Ab responses in serum capable of binding BSA conjugated E-416-433 were evident (FIG. 18A,B). The BSA-peptide conjugate is used for measuring binding to preclude detection of Abs to KLH. [Note that the requirement for mimicry of the native 421-433 CD4BS sequence by the immunogen and the probe used for measuring binding are not the same. Induced-fit mechanisms of binding shown in
In addition,
The following KLH-conjugated immunogens were also tested: E-421-433, the 421-433 peptide with a C terminal phosphonate; and Es-421-433, the 421-433 peptide with phosphonates at the side chains of Lys421 and Lys432 (
In the present invention, improved MAbs were prepared by screening 1125 splenocyte hybridomas from two mice immunized with KLH-E-416-433. Seven IgM MAbs and 13 IgG MAbs with specific binding activity for BSA conjugated E-416-433 were identified.
A subset of the MAbs (10 of 20 tested; 2 IgMs and 8 IgGs) neutralized the subtype C clinical HIV isolate ZA009 (IC50 0.1-16.5 μg/ml). IgM clones with neutralizing activity are: 1F4 and 2G9. IgG clones with neutralizing activity are: 4B2-F8, 4F6-G11, 4H12-F2, 5B5-F6, 5D3-C10, 7E2-H7, 9F3-A7 and 11G8-H4.
Broad HIV neutralizing activity was confirmed (see Table 3).
Specificity for 421-433 CD4BS sequence was shown by inhibition of MAb 2G9 binding to immobilized BSA conjugated BSA-E-416-433 in the presence of soluble full-length gp120, Bt-E-416-433 and sCD4 but not by control Sh416-433 with shuffled sequence or the irrelevant protein ovalbumin (
Molecular Features of Anti-E-416-433 Monoclonal ABS.
The VH and VL domains of the following IgM clones with specific BSA-E-416-433 binding activity were sequenced: clones 1F4, 2G2, 2G9, 2C11, H10, C11 and G12. (see sequences below). Most V domains contain few or no somatic mutations. Sequence diversification due to V-D-J and V-J recombination processes is evident.
The VH and VL domains of the following IgG clones with specific BSA-E-416-433 binding activity and HIV neutralizing activity were sequenced: clones 4B2-F8 and 9F3-A7. Both Abs shared the same VL and VH germline genes (respectively, 8-21 VK8 gene and J558.51 VH1 gene). The V domains of both MAbs contained extensive somatic mutations. The VL domains of both clones were poorly mutated (replacement/silent mutation ratios, the R/S ratios, for IgG 4B2-F8 VL and IgG 9F3-A7 domain were 1/1 and 2/1, respectively). In contrast, the VH domains were more mutated. The R/S ratios for IgG 4B2-F8 VH CDRs and FRs were respectively, 8/1 and 7/5. The R/S ratios for IgG 9F3-A7 VH CDRs and FRs were respectively, 4/1 and 3/4. The cumulative R/S ratio for FR1 and FR3 for the two neutralizing MAbs was 11/5. The expected R/S computed as in ref 76 is 8/8, suggesting immunogen-driven selection of FR mutants. The frequent FR mutations are consistent with our vaccine approach of adaptively improving the innate 421-433 CD4BS epitope recognition capability. In addition to somatic mutations in the V gene, sequence diversification of the V domains due to the V-D-J and V-J recombination processes was evident
The IgM and IgG clones displayed varying levels of BSA-E-416-433, E-120 and gp120 binding activity. Moreover, the ratios of BSA-E-416/E-gp120 and BSA-E-416-33/gp120 binding activities for the different MAb clones were divergent (respectively, by 3887-fold and 219-fold for the 20 MAbs). The widely divergent binding ratios reflects the extent to which different Ab specificities can arise due to flexibility of the 421-433 CD4BS sequence, diversity of the innate CD4BS-specific repertoire, and induced fitting of the 421-433 CD4BS into the FR-based binding site (
Amino acid sequences were deduced from the nucleotide sequence obtained by dideoxy nucleotide sequencing of PCR amplified V domain cDNA from the hybridoma cells
SKLASGVPGRFSGSGSGNSYSLTISSMEAEDVATYYCFQGSGYPYTFGGG
IHPNSGNTNYNEKFKGKATLTVGTSSSTAYVDLSSLTSEDSAVYYCARPG
ASQSISGIPSRFSGSGSGTDFTLSINSVETEDFGMYFCQQSNSWPLTFGA
VIWSGGSTDYNAAFISRLSISKDNSKSQVFFKMNSLQANDTAIYYCARTG
FAYWGRGTLVTVS
STSNLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCHQYHRSPRTFG
IRNKANGYTTEYSASVKGRFTISRDNSQSILYLQMNTLRAEDSATYYCAR
DNQSFYYAMDYWGQGTSVTVSS
TFGGGTKLEIK
IWRGGSTDYNAAFMSRLSITKDNSKSQVFFKMNSLQADDTAIYYCAKRYG
NYGGGAMDYWGQGTSVTVSS
SNLASGVPARFSGSGSGTSYSLTISRMEAEDAATYYCQQRSSYPYTFGGG
IRSKSNNYATYYADSVKDRFTISRDDSQSMLYLQMNNLKTEDTAMYYCVR
ERAGYFDVWGAGTTVTVSS
GSTLQSGIPSRFSGSGSGTDFTLTISSLEPEDFAMYYCQQHNEYPYTFGG
ISCYNGATSYNQKFKGKATFTVDTSSSTAYMQFNSLTSEDSAVYYCARGG
TTVVATGKYAMDYWGQGTSVTVSS
IWSGGSTDYNAAFISRLSISKDNSKSQVFFKMNSLQANDTAIYYCARNKD
YGSSYDYYAMDYWGQGTSVTVSS
WTFGGGTKLEIK
IYPGSGGTAYNQKFKGKATLTADKSSSTAYMELSSLTSEDSAVYYCTKFR
FSSFAMDYWGQGTSVTVSS
WTFGGGTKLEIK
FHPGSGGGAYSQKFKGKATLIADKSSSIAYMEVISLTSEDSAVYYCTRFR
YSSFAMVYWGQGTSVTVSS
Covalent and Catalytic Activity.
Covalent immunization comes with the bonus of electrophile-driven strengthening of Ab nucleophilic reactivity.5,13,57 Enhanced nucleophilicity improves HIV inactivation as follows (
Taken together, the results indicate that KLH-E-416-433 immunization favors the recruitment and adaptive improvement of preimmune catalytic activity directed to the 421-433 CD4BS sequence.
Induction of E-416-433 and E-gp120 Binding ABS in Monkeys.
We immunized 3 rhesus macaques of Indian origin with KLH-E-416-433 and 3 macaques with control KLH (see example in
To assess whether improvement in Ab activity is feasible, we immunized the three KLH-E-416-433 immunized monkeys further with E-gp120. The booster E-gp120 immunization resulted in maintenance of a steady binding activity for BSA-E-416-433 and increased E-120 binding titers (
No E-416-433 or E-gp120 IgG binding titer was observed upon control KLH immunization of three monkeys. These monkeys received three subsequent E-gp120 administrations to determine whether E-gp120 alone induces a useful response. The E-gp120 binding titers in the serum IgG fraction were 26,000-87,000 (
Induction of HIV Neutralizing Abs in Monkeys.
We measured the neutralizing activity of monkeys immunized with KLH-E-416-433 alone using primary isolates subtype C, CCR5 dependent strain 97ZA009 and PHA-stimulated human PBMCs as hosts. Only low-level neutralizing activity was observed after KLH-E-416-433 immunization (see example
We also measured the neutralizing activity of our monkeys immunized sequentially with KLH-E-416-433 followed by E-gp120. Sera from all three sequentially-immunized monkeys neutralized the subtype C, CCR5 dependent strain 97ZA009. The serum Abs neutralized genetically diverse strains drawn from subtype A, B, C, D, and AE with co-receptor CCR5 and CCR4 dependency (Table 4). We also verified that E-gp120 alone induces neutralizing Abs (
Validating HIV Neutralization In Vivo.
The simian-human immunodeficiency virus (SHIV)-macaque model is a frequent test of HIV therapies/vaccines.70 Macaques clear SHIV rapidly, but a peak of viremia is evident at 1-2 weeks. The MAb 3A5 raised by immunization with E-120 with specificity for the 421-433 CD4BS sequence,12 neutralized SHIVSF162P3 strain with potency comparable to HIV (IC50˜3 μg/mL;
In vivo proof-of-principle for protection against viral challenge in monkeys is a significant milestone in developing an HIV vaccine. SHIV strains infect rhesus monkeys. Desirable features of an effective vaccine candidate candidate are: (a) Induce Abs that neutralize diverse HIV strains in tissue culture; (b) Protect against infection contracted by vaginal SHIV administration; (c) Induce B cell memory that can be amplified by subsequent exposure to SHIV.
Protection against SHIV. E-120, KLH-E-416-433 or another electrophilic immunogen combined with a suitable adjuvant is used to induce broadly neutralizing polyclonal Ab responses. For example, one group of 8 female monkeys receives IE-gp120 in adjuvant at 2-week intervals (Experiment A,
Based on the infection kinetics and HIV amounts in semen, it has been suggested that neutralizing Ab titers of ˜1:40 may be sufficient to prevent transmission.77,78 This corresponds to an observed neutralization titer of ˜1:4 in diluted CVLF, if it is assumed that the vaginal lining fluid is diluted by 10-fold or more in the lavage procedure.
Seven days after the requisite Ab titers have been reached, the monkeys are challenged vaginally with SHIVSF162P3 (see Methods). This is a hybrid virus containing env, tat, rev and vpu genes of the CCR5-dependent subtype B strain HIV strain SF162 cloned into the SIVmac239 genome that is reliably transmitted by the mucosal route.79 Most sexually transmitted HIV infections world-wide occur by the vaginal route. The rectal transmission route may also be tested in this model. SHIVSF162P3 is a comparatively ‘difficult-to-neutralize’ strain for most anti-HIV Abs.8° Abs to the 421-433 CD4BS sequence neutralize SHIVSF162P3 (
SHIV/HIV Neutralization.
It is important to assess possible escape mutations and utility of our vaccine approach to diverse HIV strains found across the world. Initially, SHIVSF162P3 and the difficult-to-neutralize subtype C, CCR-5 dependent clinical HIV isolate ZA009 are tested for neutralization by unfractionated sera and CVLF samples from IE-gp120 immunized animals. Samples obtained before and after challenge with SHIV along with control preimmune serum and CVLF are tested. Neutralization is quantified using PBMCs from non-infected humans as described.44 In repeat assays, purified IgG, IgA, IgM and SIgA are tested. In some assays, monocyte-derived macrophages are used as hosts. Nonspecific cytotoxicity is analyzed by Ab treatment of PBMCs and vital staining.
In addition to subtype B SHIVSF162P3 strain, neutralization of genetically diverse clinical HIV isolates is measured using serum and CVLF samples. At least 4-6 clinical HIV isolates from each of subtypes A, B, C, D, E (AE env recombination) with V domains that are highly divergent in sequence are studied, including coreceptor R5-, X4- and R5X4-dependent strains. Panels of virus strains with varying resistance to Abs and known 416-433 sequences have been assembled.2,12 Residues 421-433 are largely conserved across Group M HIV-1 strains. To judge the possibility of viral escape, attention is given to Ab neutralization of strains with sequence differences at positions without effect on gp120-CD4 binding versus positions important for the binding. Potential emergence of escape mutations is tested by coculturing PBMCs infected chronically with two virus strains (SHIVSF162P3, ZA009) with the anti-IE-gp120 serum over 10 passages for 70 days. At each passage, the virus in the culture supernatants is titered using a fresh batch of PBMCs. The sequence of the gp120 gene in supernatants displaying detectable infectivity is determined to identify the amino acids residues permitting development of resistance to the Ab.
To quantify the contributions of reversible binding and catalysis in HIV neutralization, the neutralization assay (strain ZA009) are done in the presence of the E-Hapten 1 and control Hapten 2. E-Hapten 1 permits reversible binding but inhibits Ab catalysis. Hapten 2 is the control phosphonic acid devoid of inhibitory activity. Reduced neutralization in the presence of E-Hapten 1 suggests that catalysis enhances Ab neutralizing activity.
Ab neutralization of genetically diverse HIV strains and no or minimal viral escape mutants can be interpreted to suggest the feasibility of vaccination around the world, as opposed to relying on constantly changing vaccine formulations for viral subtypes or new virus strains (e.g., influenza virus vaccines are reformulated as new viral variants evolve). The CD4BS region is essential for infection. This provides a selective pressure against viral escape from anti-CD4BS Abs.
Ab Characterization.
Ab binding of IE-gp120 and BSA-conjugated E-416-433 is measured by ELISA. IgG, IgA and IgM titers will be measured separately to confirm that there is no class restriction. Increasing specific binding of BSA-E-416-433 induced over the course of KLH-E-416-433 immunization indicates that an adaptive Ab response has occurred. The specificity of immobilized BSA-E-416-433 binding by plasma and CVLF Abs is confirmed by competitive inhibition with soluble CD4 as described.2 Cleavage of purified gp120 is measured using electrophoretically homogeneous secretory IgA (SIgA) from CVLF and IgA, IgG and IgM from sera after purification by affinity chromatography (see Methods). The catalysis data are confirmed by showing retention of the activity in Ab preparations prepared by denaturing gel filtration (to remove noncovalently associated trace contaminant) 9,82 Peptide bonds cleaved by Abs are identified from the mass distribution of gp120 fragments on electrophoresis gels and by N-terminal sequencing 9 Inhibition of catalysis by sCD4 but not irrelevant proteins is quantified to confirm noncovalent CD4BS recognition by the catalytic Abs. Apparent Km (approximate measure of KO and apparent maximal velocity/unit Ab mass (Vmax) are computed from rates observed at varying gp120 concentrations.9 In addition to purified gp120, the trimeric gp120 expressed by intact HIV particles is employed as substrate to establish binding and catalytic hydrolysis attributable to recognition of the native 421-433 CD4BS sequence (see
Vaccine Toxicity/Ab Cross-Reactivity.
The control and IE-gp120 immunized animals are monitored as follow: (i) Evaluation of immunogen administration site for irritation/inflammation; (ii) Behavioral examination; (iii) Physical examinations (body weight, body temperature, blood pressure); (iv) Blood/urine cellular and chemistry profiles; (v) At autopsy, gross observation, organ weights and histopathology evaluation of various organs. Blood coagulation occurs by a series of serine protease reactions. To rule out nonspecific interference with blood coagulation, we monitor the activated partial thromboplastin time and prothrombin time ratio as reporters of the intrinsic and extrinsic coagulation pathway, respectively. Ab cross-reactions with human tissues is studied to assess the possibility of autoimmune damage. As an example, the following de-identified tissues obtained at autopsy from humans without HIV infection are tested: heart, lung, kidney, liver, stomach, small and large intestine, spleen and brain. Cryostat tissue sections treated with monkey anti-IE-gp120 IgG or control monkey IgG from animals immunized with adjuvant alone are stained with peroxidase-conjugated Abs to monkey IgG and examined by microscopy. Equivalent tissue staining by the anti-E-416-433 IgG and control IgG suggests lack of cross-reactions with human proteins. The Ab-treated sections are also be stained with peroxidase-conjugated Abs to commonly expressed tissue antigens such as actin, tubulin, myosin, vimetin and CD56. Unimpaired staining of these antigens suggests lack of indiscriminate polypeptide digestion by the Abs.
SHIV-Induced Protective Memory Response.
An important feature of vaccines is the induction of B cell memory enabling a subsequent protective Ab response upon contact with microbe. Therefore, SHIV challenge is also performed after the plasma and CVLF neutralizing Ab titers have declined to low levels (<10% of peak Ab response in Experiment B,
For peptide immunogens, the carrier protein is an important factor governing the quantity and quality of the Ab response. In addition, the adjuvant is important to maximize the Ab response both for peptide and protein immunogens. Alternate carriers and adjuvants are tested using monkeys or rabbits to optimize the vaccine formulation.
Carrier/Adjuvant effects.
Rabbits offer a well-established animal model to study candidate vaccines, affording sufficient production of mucosal and systemic Abs for detailed analysis of functional properties. In the examples shown here, New Zealand White female rabbits are used (n=4/group). Inducing mucosal immunity is important to prevent HIV transmission. Inducing systemic immunity is important for controlling spread of infection.
As an example, immunizations are done by alternate intranasal and intramuscular KLH-E-416-433 administration at 2 week intervals to induce strong mucosal as well as systemic Ab responses as described previously.83 Intranasal immunization results in broad and specific B cell immunity expressed at distant mucosal sites, including the genitals and gastro-intestinal tract.84 B cell responses to peptides depend in part on generating antigen-specific helper T cells to TH-epitopes expressed on the carrier protein. Costimulatory helper T cell signaling helps drive somatic diversification of the Ab V domains and Ab class-switching over the course of B cell maturation.85
In addition to KLH, other protein carriers that support folding of the 421-433 CD4BS epitope in a near-native conformation can be used, for example, tetanus toxoid and CD40 ligand (Table 5; 300 μg peptide equivalents/rabbit). The KLH-E-416-433 conjugate contains ˜2000 copies of E-416-433 linked via an N terminal Cys located in the peptide to Lys side chains of KLH. Similar conjugates of E-416-433 to tetanus toxoid (TT) and CD40 ligand (CD40L) are prepared. Tetanus toxin and CD40L contain, respectively, 107 and 16 Lys residues. Preparation of TT entails an aldehyde reaction with amines, but sufficient underivatized Lys residues are available for the conjugation reaction. TT is often used in conjugate vaccines involving poorly immunogenic polysaccharide antigens, and it is approved for human use (e.g., ACTHIB, a TT conjugated-polyribosylribitol phosphate for H. influenza serotype b infection). Moreover, pre-existing memory acquired by childhood tetanus vaccination may help sustain the B cell response to the 421-433 epitope. Improved B cell responses due to pre-existing anti-carrier protein memory has been observed previously.86 As an example, the TT-E-416-433 conjugate is tested in separate rabbit groups without and with prior TT immunization to assess the facilitatory role of anti-carrier memory (one IN and one IM administration at 2 wk intervals). Inclusion of CD40L as a carrier protein is shown to improve B cell Ab synthesis by virtue of the co-stimulatory signal generated upon CD40L binding to CD40 expressed on activated T cells.87 To minimize possible loss of CD40L functional activity, we can adjust the number of E-416-433 copies to 3-5/CD40L molecule. The mucosal and systemic adjuvants can be LTm and RIBI, as these adjuvants were verified to support the desired Ab response.
Adjuvant can enhance the Ab response by several log orders. Adjuvants improve the immune response by virtue of various physical and chemical factors, including: improved immunogen bioavailability mediated by adsorptive effects; provision of a hydrophobic environment that improves immunogen interactions with cell surface receptors; and stimulation of innate immunity pathways. Adjuvants can activate specific toll-like receptors and other receptors on antigen-presenting cells and T cells, thereby inducing release of cytokines and expression of costimulatory molecule. Adjuvants that present the immunogen within small-sized physical units generally offer improved responses.
As examples, in addition to Ribi and LTm, various systemic and mucosal adjuvants can be used (Table 5). Aluminum hydroxide (alum) is approved for human use. This adjuvant adsorbs immunogens and then releases it slowly. In addition, it was recently found to activate innate immunity via the nucleotide-binding domain leucine-rich repeat-containing protein 3 ‘inflammosome’ pathway.88 W805EC is an oil-in-water nanoparticle emulsion composed of cetyl pyridinium chloride, soybean oil, Tween 80 and ethanol with mean droplet size <400 nm diameter. It is reported to facilitate induction of Abs to viral and bacterial proteins with titers in the 1:106 range.89 Cholera toxin A1 subunit linked to the DD Ig-binding domains of staphylococcal protein A, is a non-toxic TH1/TH2 adjuvant that enhances mucosal Ab responses to HIV and other immunogens. CpG ODN is a toll-like receptor 9 agonist that favors TH1 responses by actions on dendritic cells and B cells.9°
Characterization of Abs.
The desired Ab activities are tested in sera, CVLF and fecal pellet extracts collected from rabbits using well-documented procedures91-93 before and 1 wk after each immunization. The Ab activities to be tested are essentially as described in Example 1 Activities that are tested are: (A) Potency with which the Abs neutralize genetically diverse HIV strains; (B) Binding of BSA-E-416-433 peptide; and (C) Catalytic hydrolysis of gp120.
Taken together, the studies are designed to identify the carrier protein and adjuvant supporting enhanced production of neutralizing Abs to the 421-433 CD4BS epitope.
Our results predict a variety of ways to obtain improved immunogens expressing the 421-433 CD4BS sequence in a conformation that better resembles the native conformation of this sequence on the viral surface. Such improved immunogens are predicted to induce the synthesis of HIV neutralizing Abs at greater magnitude and with greater potency than current generation vaccine candidates.
Structure of Alternate E-Vaccine Candidates.
The prototype KLH-E-416-433 vaccine and E-120 contain the 421-433 CD4BS epitope recognized by the FRs of BCRs found in the innate Ab repertoire without prior exposure to HIV. Studies on the secreted Abs from non-infected humans have indicated that the innate BCRs recognize the native 421-433 CD4BS sequence of the virus sufficiently to neutralize HIV (
As examples, five classes of immunogens described below can be tested for improved 421-433 CD4BS conformation.
For each class of test immunogens, the structure of the electrophile can also be varied to provide optimum reaction with nucleophiles expressed by the Abs on B cells. For example, phosphonate monoesters, aldehydic or keto compounds, dicarbonyl compounds, lipid peroxidation products, boronate compounds and vanadate compounds can be employed as alternate electrophiles. The chemical constitution and length of the linker can also be varied. Examples of dicarbonyl electrophiles included the Advanced Glycation Endproducts obtained by the reaction of sugars with proteins. Another example of a protein electrophile is the reaction product of 4-hydroxy-2-nonenal with protein groups such as the nucleophilic side chains of Lys residues (
As an example, in the phenylphosphonate structure with E-416-433 in
In every case, an appropriate carrier protein and adjuvant are used to prepare the candidate vaccine formulation using methods described in Example 8.
The five classes of novel immunogens are:
A. Degenerate E-416-433 (degE-416-433). The KLH-E-416-433 immunogen contains the consensus epitope sequence. Table 1 shows the extent to which individual amino acids of the epitope are conserved in 1699 Group M HIV strains available in the databanks Abs to the 421-433 CD4BS epitope neutralized all HIV strains potently, but the neutralizing potencies were superior by 2-3 log orders for across the panel of strains.2 The variable potency may derive in part to epitope sequence divergences. Consequently, a degenerate degE-421-433 immunogen that includes peptides expressing greater sequence identity with the individual epitope sequences of diverse HIV strains can induce Abs with more consistent neutralizing activity across the strains. Conservation of the epitope sequence is >95% at all but 4 of its 18 positions. As examples, degeneracies at these 4 positions can be introduced with the objective of including peptide species in which the rare sequence divergences are better represented. An example degE-416-433 immunogen is composed of 24 peptides with the following sequence: L-P/Q-C-R-I-K-Q-I-I-N/R-M/R-W-Q-E/R/G-V-G-K-A. This immunogen contains E-peptide species with the amino acids at individual positions encompassing >95% of Group M HIV strains.
B. Rigidified E-416-433. Short peptides are flexible and can fold into alternate conformations.52,94 Induced-fit binding mechanisms can force KLH-E-416-433 bound to BCRs into a conformation deviating from the native CD4BS conformation expressed on the HIV surface (
As an example, the hydrocarbon stapling' method95 can be used to produce rigidified E-416-433 variants with stabilized α-helix or β-sheet structures, βE-416-433 and (3E-416-433.
An additional example of an improved immunogen likely due to a rigidification effect is evident from comparison of the E-416-433 peptide with its non-electrophilic peptide 416-433 counterpart devoid of the electrophilic phosphonate groups.
C. CD4BS expansion. Lengthening the 416-433 epitope further can provide additional stabilization interactions in the folding of the synthetic peptide, resulting in improved mimicry of the native CD4BS. For example, we observed improved CD4 binding by E-414-439, a CD4BS mimetic containing the additional 414-415 and 434-439 residues drawn from the Group M consensus gp120 sequence (
Further lengthening of the peptide flanks of the 421-433 CD4BS sequence can improve its conformation. However, the sequence of gp120 in group M HIV strains is increasingly variable as the flanks are lengthened. To avoid excessive structural diversity, it is advisable to limit the immunogen length to about 49 amino acids corresponding to the consensus sequence of gp120 surrounding the 421-433 CD4BS sequence (numbered residues 406-459 according to strain HXB2 numbering system employed in the present invention; note that the HXB2 strain contains a 5 residues insert not present in the consensus sequence). The consensus sequence corresponding to group M HIV-1 gp120 amino acid position 406-459 is:
The means for expanding the epitope usefully are not limited to the contiguous gp120 regions flanking the 416-433 region. Spatially remote amino acids or peptide regions can be included on the two flanks to improve the vaccine quality. For example, the outer domain residues 368-370 are thought to be components of the CD4BS along with the 421-433 sequence. A peptide sequence that spans residues 368-370, for example synthetic peptide 365-371, may be included on the N or C terminal flank of E-416-433. A linker can be placed between residues 365-371 and residues 416-433 to approximate the distance between these regions on the surface of gp120. Such expanded CD4BS immunogens may be expected to improve the induction of neutralizing Abs to HIV.
D. E-mimotopes. In the course of studies of synthetic peptide 416-433 in which individual amino acids were replaced by an irrelevant residues (Alanine), we noticed that certain replacements result in improved peptide binding to known neutralizing Abs directed to the 421-433 CD4BS sequence. Concordant but modest improvements were evident for binding of such mutant peptides by MAb IgM 2G9 raised by immunization with KLH-E-416-433 and IgA from long-term survivors of HIV infection. As examples, replacement of Pro417, G1n422 or Glu429 resulted in improved binding to these Abs, evident from ELISA competition studies (Table 6).
From the fortuitous improvements in binding displayed by the mutant 416-433 peptides, it can be predicted that a mimotope with conformation similar to the native 421-433 CD4BS sequence can be isolated from a random peptide library provided the appropriate selection and screening technologies are applied. It is well-known that screening of large libraries composed of peptides with random sequence can yield rare mimotopes that do not have the same linear sequence of the native epitope but mimic the conformation of the native epitope. Indeed, even small molecule analogs of the native epitope can be identified by screening of small molecule libraries. Molecular modeling of the Ab-ligand interaction can be done to guide the refinement of antigen structures that mimic the native epitope optimally, for example by introducing non-polar or polar substituents with varying bulk into the antigen structure. Libraries of peptides and small molecules are available commercially, and synthetic procedures for semi-rational improvement of the mimotope are also well-established.
Methods for displaying the random peptide library on a suitable surface followed by selection and screening of the desired mimotopes are well established. For example, the library can be displayed on the surface of M13 phage or ribosomes.98,99 In the example of a phage displayed library, selection is done using immobilized sCD4 or an immobilized neutralizing Ab to the 421-433 CD4BS sequence (for example scFv JL427). Thereafter, individual phage peptide clones with the desired binding activity are identified by screening for CD4 or Ab binding using ELISA methods.
Implementation of these procedures to the 421-433 epitope can be predicted to yield a mimotope with a conformation mimicking the native 421-433 CD4BS sequence. One or more electrophilic group can then be incorporated into the mimotope at the side chain of an appropriate amino acid, and the resultant E-mimotope can be tested as immunogen for induction of neutralizing anti-CD4BS Abs in experimental animals.
E. Binary epitope E-immunogens. An effective HIV vaccine must induce immunological memory stimulated upon contact with the virus. If deficient class-switch is the only impediment in the natural immune response, once a class-switched memory response has been induced by covalent immunization, contact with HIV should stimulate differentiation of the memory B cells into plasma cells. However, if unforeseen post-class switch immune impediments exist, then contact with the virus may not stimulate the covalent immunogen induced memory cells sufficiently. A binary E-vaccine is predicted to induce memory B cells with specificity for a second epitope in addition to the 421-433 CD4BS epitope. As the binary E-vaccine and HIV share the second epitope, the CDRs expressed by the memory cells will bind HIV, generating a stimulatory signal that overcomes down-regulatory FR binding to the viral 421-433 CD4BS sequence (
Immunogen Testing.
Immunogens are tested as in Examples 1 using groups of 4 rabbits each. As examples, the following immunogens are tested: the binary E-CD4BS-Li-Epitope2 immunogens, degenerate degE-416-433, the stapled E-immunogens, the lengthened E-immunogens and the E-mimotope immunogens. The immunogens are coupled to KLH as before and rabbits receive alternate intranasal and intramuscular immunogen administrations in LTm and Ribi, respectively. Blood, CVLF, feces and lymphoid tissues are collected for Ab studies.
The following tests are done to determine the quantity and quality of the Abs induced by the immunogens: (A) Potency of neutralization of genetically diverse HIV strains: (B) Binding to the BSA-conjugated immunogens; and (C) Catalytic hydrolysis of gp120.
Emergence of viral escape mutants is tested by coculturing infected cells with an Ab preparation. The degE-421-433 immunogen more comprehensively represents the diversity of Group M HIV epitope sequences. Abs to this immunogen may display improved breadth of neutralization across diverse HIV strains, determined by testing the panel of genetically diverse strains from various HIV subtypes.
Memory tests are conducted to compare HIV stimulation of B cell memory induced by the single epitope E-CD4BS immunogen and the binary E-CD4BS-Li-Epitope2 immunogens administered to groups of 6 rabbits each. The E-CD4BS binding titers are measured every 4 weeks after the final immunogen administration until the titer decreases to low levels. Then, a subgroup of 3 rabbits from each group receive a single booster of the appropriate E-CD4BS immunogen (positive control). The second subgroup of 3 rabbits from each group receive a booster of photochemically-inactivated intact HIV (strain ZA009). Another control group of rabbits without prior E-immunogen exposure receives only the HIV administration. One week later, the HIV neutralizing activity of Abs in the blood, CVLF and fecal extracts is measured. Rabbits that receive HIV alone are anticipated to display only low-level Abs directed to the 421-433 CD4BS epitope. Productive HIV stimulation of the E-immunogen induced memory is suggested by increased neutralizing Ab synthesis following the HIV booster.
Specific MAbs are generated to evaluate the functional consistency of individual Abs constituting polyclonal Ab preparations. Splenocytes are from a rabbit immunized with the immunogen affording the greatest neutralizing polyclonal Ab response. The rabbit myeloma 240-W derived cell line is the fusion partner.100,101 Hybridomas are screened for HIV neutralizing activity and cloned by limiting dilution. MAbs are purified by affinity chromatography (Protein G or anti-Ig columns).
The rigidified E-immunogens can induce Abs with superior neutralizing potency due to stable mimicry of the native epitope. The degenerate E-immunogen can induce Abs with more consistent neutralizing activity across diverse HIV strains found worldwide. Combined immunizations using the single epitope E-immunogen and the binary E-immunogens may help induce B cell memory that is more readily stimulated upon contact with HIV itself. The catalytic activity of the Abs is anticipated to improve neutralizing potency.
Our singular focus on the 421-433 epitope is open to the criticism that alternate epitopes may be more suitable vaccine targets. This is not the case. HIV offers very few conserved epitopes suitable for vaccine targeting. Indeed, no immunogen other than the E-immunogens are known to induce a neutralizing Ab response to the CD4BS. The superantigenic character of the CD4BS is a newly recognized challenge in HIV vaccine research. Our covalent immunization strategy is a viable solution to this challenge.
Prophylaxis
Controlling the HIV pandemic globally requires a prophylactic HIV vaccine. The E-immunogens disclosed in the present invention may be developed to prepare an effective prophylactic vaccine that is globally effective by virtue of inducing synthesis of Abs to the CD4BS that neutralize diverse Group M HIV strains found worldwide. Vaccination with an E-immunogen early in childhood can be foreseen as a way to prevent HIV infection. Adult vaccinations are also feasible. To maintain immune memory, periodic booster administrations of the E-immunogen will likely be necessary. Inducing mucosal immunity, for example by intranasal or oral immunization, is important to generate secretory IgA responses that reduce the probability of mucosal transmission of HIV. Inducing systemic immunity is important to minimize spread of infection by HIV that may breach the mucosal barrier.
Gene immunoprophylaxis using an Ab to the CD4BS that is produced systemically and locally in the vagina or rectum by means of a suitable vector, for example an adeno-associated viral vector (AAV), may also be feasible. AAV vectors providing Ab fragments over long durations of months to years have been developed. The procedure entails, for example, expression of neutralizing Ab variable domain genes cloned in a non-toxic, minimally immunogenic AAV vector in epithelial cells or muscle cells. This permits secretion of the Ab variable domains into mucosal fluid and/or blood. A suitable molecular form of the variable domains is the single chain Fv containing the VL and VH domains linked by a flexible peptide.
The foregoing immunoprophylactic strategies are anticipated to be effective at low cost and with minimal side effects. A variety of small molecule drugs that target the HIV reverse transcriptase, protease and integrase have been developed. Use of such drugs alone or as combined regimens by the sexual partners of infected humans has been proposed as a way to prevent HIV infection. However, these drugs are comparatively expensive and their routine use can induce serious side effects.
The side effects of the available small molecule drugs include diarrhea, nausea, vomiting, lipodystrophy, hyperglycemia, liver toxicity, pancreatitis, neuropathy, adverse nervous system effects (depression, suicidal ideation and paranoia) and increased rate of certain infections. Patient compliance is a problem due to the side effects.
Therapy
Combinations of the small molecules drugs are the mainstay of HIV therapy (highly effective anti-retroviral therapy, HAART). HAART reduces viral load by several orders of magnitude, sometime to undetectable levels. However, in addition to the problems of side effects noted above, about 10-20% of patients develop drug-resistant HIV strains.
Therapeutic vaccination with E-immunogens is a viable strategy to control infection if the vaccine induces sufficient production of broadly neutralizing Abs that do not permit development of viral escape mutants (Ab-resistant strains). Therapeutic vaccination can be initiated early after diagnosis of infection when the immune system is fully competent in mounting the vaccine-induced Ab response. Overall immune exhaustion in infected patients occurs only at the very advanced stage of infection, and the immune system generally remains competent over several years prior to progress of the infection to AIDS. Thus, finding a window of time sufficient to initiate therapeutic vaccination is not a problem.
Immunotherapy of HIV infection using intravenously infused catalytic Abs disclosed in the present invention is feasible. Previous therapy trials of anti-HIV Abs have not been successful because of insufficient neutralizing potency, insufficient ability to neutralize genetically diverse HIV strains and emergence of Ab-resistant strains. Targeting of the 421-433 CD4BS sequence by catalytic Abs is anticipated to minimize the problems encountered with other types of Abs.
In addition to intravenous infusion, the catalytic Abs disclosed in the present invention could be delivered by means of the AAV expression vector described above. The gene immunotherapy approach will reduce the costs of treatment.
The foregoing therapeutic vaccination and catalytic Ab approaches could be combined with HAART to maximize efficacy and reduce the requirement for toxic HAART regimens.
The HIV genome integrates into host chromosomes, giving rise to the problem of viral latency. Drugs that may address this problem are under study, for example drugs that induce virus packaging from the latent viral genomes by activating certain cell surface receptors. The therapeutic vaccination and catalytic Ab approaches could be combined with such drugs to address the problem of HIV latency.
Immunogens/Adjuvants.
Synthesis of peptides, E-peptides and E-gp120 has been described.2,3,6,57 E-414-439 and 416-433 peptides contain the consensus subtype B epitope sequence. They are constructed by solid phase synthesis with site-directed acylation at Lys side chains using the N-hydroxysuccinimide ester of the diphenylphosphonate substituent. For binary epitope E-peptide synthesis, epitope 2 is attached to a Cys residue at the N terminus of the single epitope E-vaccine using a Gly/Ser linker with an N-terminal γ-maleimidobutyryl group. Linker length approximates the distance between the two epitopes measured on the surface of the gp120 crystal structure (PDB 2B4C) using Accelrys DS vizualizer 2.0. Epitope 2 is composed of the indicated gp120 residues. For synthesis of rigidified E-416-433 analogs, the following protected linear peptides containing two α-alkenyl alanine residues are prepared by the solid-phase method: αE-416-433 precursor containing R-2-(4′-pentenyl)alanine residues at positions 418/422, and βE-416-433 precursor containing R-2-(2′-ppropenyl)alanine and R-2-(4′-pentenyl)alanine at positions 423/C-terminus. The standard 9-fluorenylmethoxy protection scheme is used except that Lys41 1 and Lys432 are protected with the 4-methyltrityl group. The protected peptide resin is treated with bis(tricyclohexylphosphine)benzylidine ruthenium (IV) dichloride under anaerobic conditions. Introduction of phosphonate groups to metathesized peptide precursors and removal of protecting groups is done in the same manner as E-416-433. Degenerate degE-416-433 peptides are synthesized by the “portioning-mixing” method. The peptide-resin is divided into the number of residues to be coupled, and coupling conducted individually. After complete acylation is confirmed (ninhydrin test), the peptide resin is pooled for additional synthesis cycles. All compounds are HPLC purified and their structures verified by mass spectroscopy (MS). The presence of all 24 expected components in the degenerate E-416-433 is verified by MS. Peptides are conjugated to Lys residues of KLH, BSA CD40L or tetanus toxoid by means of a Cys residue using a heterobifunctional reagent.44 The conjugation reaction is measured from consumption of—SH groups. Recombinant full length gp120 is from Immunodiagnostics, Inc or Protein Scineces, Inc. Proteins are biotinylated at Lys residues.6W805EC is prepared as described.89 LTm is provided by Dr Clements.14 CpG ODN2007 (TCGTCGTTGTCGTTTTGTCGTT) is prepared by routine synthesis.
Monkey Procedures.
Cycling female Indian rhesus macaques (Macaca mulatta; 4.8-6.1 kg; negative for type D retrovirus, SIV and simian T-lymphotropic virus) are studied. Intranasal immunizations is done by immunogen instillation into each nare (0.05 ml) with LTm (0.25 mg) as adjuvant. Intramuscular immunizations is done in the upper thigh using RIBI as adjuvant. Thirty days before SHIV challenge, 30 mg Depo-Provera is administered to allow more efficient infection.102 To eliminate vaginosis as a factor in the infection process, culture swabs are obtained from all animals at least 3 weeks prior to virus challenge for bacterial identification and antibiotic sensitivity testing. Animals are treated with an oral antibiotic (Enrofloxacin, 5 mg/kg daily) for 7 days. Newly expanded SHIV162P3 stock prepared by in vivo passage is used as the challenge virus. Typically, the stock has 30 ng/ml of SIV p2′7, TCID50 value of 5200/ml in rhesus PBMC and in vivo MID50 of 1:69.6 for vaginal transmission in Depo Provera-treated rhesus macaques. Virus challenge is done by instilling SHIVSF162P3 into the vagina. Blood is collected periodically from the femoral artery. CVLF is collected by recovery of 3 ml PBS instilled into the vagina. The animal is anesthetized using ketamine-HCl (10 mg/kg)/Domitor (0.03 mg/kg). Plasma viral RNA load is quantified by a real-time nucleic-acid-sequence-based amplification assay (NASBA). Viral RNA/proviral DNA load in PBMC, spleen, inguinal and axillary lymph nodes, mesenteric lymph node and intestinal lamina propria after euthanasia is quantified by NASBA and real-time PCR.103,104 CD4+ T-cell counts in blood is assayed by flow cytometry.104,105 Immunogen administration sites are evaluated pre-inoculation and daily for 3 days post-inoculation for erythema, ulceration and edema. Behavioral examinations consist of monitoring posture, mobility and food/water consumption. Physical examinations are performed weekly. Blood samples are subjected to routine CBC (includes WBC differential), hematocrit; serum chemistry with SMAC (complete chemistry/electrolytes), creatinine, alkaline phosphatase and aspartic serum transferase. Complete urine analyses are performed. Autopsy includes gross observation, organ weights and histopathological examination of heart, lung, kidney, liver, stomach and intestine.
Rabbit Immunization/Sample Collection.
New Zealand White female rabbits (n=4 per group) are immunized 4 times every 2-weeks. Intranasal immunizations consists of immunogen instillation into each nostril (0.05 ml) using LTm (0.25 mg), W805EC (final 20% (v/v)),89 CTA1-DD (0.25 mg)106 or CpG (20 μg)107 as adjuvant. Intramuscular immunizations are done using RIBI (1:1; v/v), aluminum hydroxide (2 mg) 108 or W805EC (final 20% (v/v)) 89 as adjuvant. Blood is collected from the ear. CVLF is collected following instillation of PBS (1 ml) into the vagina. Fecal pellets collected over one day are homogenized in PBS (0.1 g/ml) and the supernatant containing Abs are recovered by centrifugation.
Immunochemical Ab Assays.
Secreted IgM, IgA and IgG from serum, CVLF and fecal extracts are purified to electrophoretic homogeneity using columns of immobilized anti-rabbit IgM/IgA, anti-monkey IgM/IgA or Protein G. Rabbit MAbs are prepared using the myeloma 240-W derived cell line as fusion partner. Endotoxin levels in Abs are estimated by the Limulus amebocyte lysate test using the Endosafe-PTS instrument and FDA-approved cartridges. Trace amounts of endotoxin were removed in our previous studies using anion exchange cartridges to ensure that endotoxin does not interfere in the neutralization assays.2,12 Ab binding activity is tested by ELISA using immobilized antigens.6,44 Soluble competitor peptides are used to show specificity. Photochemical psoralen inactivation of HIV has been described.109 To measure binding to HIV, immune complexes of the virus are trapped on anti-Ig affinity chromatography columns and the bound fraction eluted with pH 2.7 buffer is treated with Triton X-100 to lyse HIV particles, followed by p24 ELISA.13 Catalytic activity is tested using purified Abs and biotinylated gp120 or irrelevant proteins by an electrophoresis assay.6 Appearance of small mass products and depletion of the intact gp120 band indicate cleavage. We also use a recently-standardized assay to measure cleavage of viral gp120 using as substrate 35S-Met labeled HIV (˜105 cpm; obtained at the void volume of a Sephacryl-100 gel filtration column). After incubation with Abs, HIV is lysed and the intact gp120/immunoreactive fragments immunoprecipated with pooled anti-HIV Abs from HIV-infected humans are analyzed by SDS-gel electrophoresis and densitometry. Kinetic parameters (Km, Vmax) are computed from rate data at increasing substrate concentration by fitting to the Michaelis-Menten-Henri equation V=Vmax*[S]/(Km+[S]).6 Scissile bonds are identified by N-terminal sequencing of gp120 fragments separated by electrophoresis and blotted on PVDF membranes.9 Viral gp120 scissile bonds are determined by a sensitive radiosequencing method using HIV labeled in tissue culture with a mixture of radioactive amino acids. As the gp120 sequence is known, the cleavage site can be deduced from the cycle in which the radiolabeled PTH-derivitized amino acids elute.11° A human tissue bank is available for Ab cross-reaction studies. Tissue cryostat sections treated with the test Ab are stained with peroxidase-conjugated Ab to macaque IgG. Controls include the second Ab alone. Fc-receptor block reagent (Pharmingen) is used to eliminate binding to Fc receptors. Ab binding is quantified by computer-assisted microscopy expressed as pixels/unit area of the section.
Neutralization Assays.
Neutralization of clinical HIV isolates obtained from the NIH AIDS Reagent Repository is tested using phytohemagglutinin-activated human PBMCs pooled from 4-12 donors.44 The virus is incubated with the test Ab sample in quadruplicate to ensure reliability. The reaction mixtures are added to PBMCs in 96-well plates. Infection is monitored using p24 enzyme-immunoassay kits. Additional confirmatory assays are done using monocyte-derived macrophages as hosts cells.111 Neutralization of pseudovirions expressing various env genes is also measured by a luciferase assay using the TZM-b1 host cell line.61 Emergence of escape mutant in vitro is tested as follows. The stock HIV strain ZA009 is used to infect PBMC in the presence of the Ab. Infection proceeds overnight and cells are washed and resuspended in RPMI media containing the Ab. After 7 days the cell-free supernatant is harvested and used to infect a new aliquot of stimulated PBMCs (second passage). The virus is passaged 10 times with a gradual (2-fold) increase in Ab concentration with each passage. A virus aliquot is saved at each passage for sequencing.112 Viral supernatants are titered at each passage using a fresh batch of PBMCs. The gp120 gene will sequenced following RT-PCR as described.113
Each of the patent applications, patents and other publications cited herein is incorporated by reference in its entirety.
Although the foregoing description is directed to the preferred embodiments of the invention, it is noted that other variations and modifications will be apparent to those skilled in the art, and may be made without departing from the spirit or scope of the invention. Moreover, features described in connection with one embodiment of the invention may be used in conjunction with other embodiments, even if not explicitly stated above.
This application claims the benefit of U.S. provisional patent application Ser. No. 61/365,139 filed Jul. 16, 2010, which is hereby incorporated by reference in its entirety.
Pursuant to 35 U.S.C. §202(c) it is acknowledged that the U.S. Government has certain rights in the invention described herein, which was made in part with funds from the National Institutes of Health grants R01 AI067020; R01 AI058865.
| Number | Date | Country | |
|---|---|---|---|
| 61365139 | Jul 2010 | US |
