Patent Application
20130164316
The present invention relates generally to HIV-1 and, in particular, to immunogens that elicit broadly neutralizing antibodies against HIV-1 subtype C envelope glycoproteins, to compositions comprising same, and to methods of inducing the production of such antibodies in a subject.
The ability to elicit broadly cross-reactive neutralizing antibodies (Nabs) is an important goal for HIV-1 vaccines [1,2]. HIV-1 has nine genetically related lineages (subtypes A-K), and at a minimum at least one Glade should be effectively targeted in an HIV vaccine for that vaccine to be useful in a part of the world where a single subtype dominates the epidemic [3]. Nabs capable of targeting subtype C variants of the virus would be particularly useful, since subtype C accounts for approximately 50% of all HIV-1 infections world-wide [4], and the HIV epidemic in large regions of Southern Africa and in India is almost completely dominated by subtype C infections. Accordingly, there is a need to focus on subtype C antibody responses in natural infection. Efforts to generate cross-reactive Nabs have met with limited success, and novel approaches are urgently needed [1,5]. The potent cross-reactive neutralizing activity that is seen with a subset of human monoclonal Abs [6,7,8,9,10] and serum samples from HIV-1-infected individuals [11,12,13,14,15,16] is evidence that improvements in immunogen design may be possible.
Insights into how to make such improvements are being sought by studying autologous and heterologous Nabs in HIV-1-positive serum samples. Recent results indicate that epitopes in and around the CD4 binding site of gpl 20 comprise key targets for broadly neutralizing HIV-1-positive sera and that other key targets remain to be identified that play a substantial role [13,14,15]. Studies of autologous virus neutralization have shown that the response targets multiple regions of gp120, most notably epitopes in V1/V2 [17] and epitopes that require an interaction between C3 and V4 [17,18,19,20], A recent study identified 19 amino acid signatures in gp120 and gp41 that associated with the neutralization susceptibility of a multi-subtype panel of viruses [21]. Walker et al. have recently found two mAbs that bind to conformation determinants of HIV-1 Env and broadly neutralize about two-thirds of viruses tested [22].
Previous studies of the autologous and heterologous Nab response in HIV-1 infection have utilized one or limited number of representative env genes from each individual to characterize neutralization susceptibility [23,24,25,26]. Since env is highly variable in chronic HIV-1 infection (CHI) and because minor sequence changes can affect the biological function and antigenicity of the envelope glycoproteins [27,28,29,30,31,32], the study of a single env gene from each infected individual provides only a minimal representation of viral populations in vivo. Thus, depending on the design of the study, key information about neutralization epitopes may be missed under these conditions.
Another limitation of previous studies is that most have relied on a traditional bulk PCR methodology for Env cloning rather than single genome amplification (SGA) [23,24,25,26,33]. Viral sequences from the quasispecies population obtained by bulk PCR can result in artificial recombination and re-sampling as well as in nucleoside misincorporation by low fidelity Taq polymerase [34,35,36]. The SGA methodology makes it possible to obtain bona fide viral genomes from the infected individual [33,36,37,38]. Because viral sequences obtained by SGA more accurately reflect what is present in vivo [37], they can be used to better characterize viral gene functions.
The present study results, at least in part, from the use of SGA and a novel promoter PCR method to express functional Envs in a high throughput format [38] in order to explore the question of whether there are common neutralization signatures evident in Glade C viral sequences that associate with broad Nab responses. Multiple env genes from each of 37 HIV-1 infected individuals were obtained and characterized with respect to their infectivity and their susceptibility to neutralization by autologous and heterologous plasma samples. A signature sequence was found near the fourth variable region (V4) of gp120 that was associated with potent Nab responses in subtype C HIV-1-infected individuals.
In general, the present invention relates to HIV-1. More specifically, the invention relates to immunogens that elicit broadly neutralizing antibodies against HIV-1 subtype C envelope glycoproteins, and to compositions comprising same. The invention further relates to methods of inducing the production of broadly neutralizing antibodies in a subject.
Objects and advantages of the present invention will be clear from the description that follows.
The patent application file contains at least one drawing executed in color. Copies of this patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
An increase in knowledge of the molecular and antigenic structure of the HIV-1 envelope glycoproteins (Env) has yielded important new insights for vaccine design but translating this information to an immunogen that elicits broadly neutralizing antibodies has been difficult. The present invention is based, at least in part, from the use of SGA and a novel promoter PCR method to express functional Envs in a high throughput format [38] in order to explore the question of whether there are common neutralization signatures evident in Glade C viral sequences that associate with broad Nab responses. As described in the Example that follows, the neutralization properties of serum antibodies for autologous and heterologous multiple authentic and functional HIV-1 env genes in HIV-1 Glade C quasispecies from 34 chronically infected individuals were characterized. A total of 474 full-length HIV-1 env genes (5-23 per subject) were obtained by single genome amplification. One-third of these individuals harbored populations of clonally expanded viruses with identical or similar genetic sequences that accounted for 9%-38% of the total virus population. Functional analysis of these env genes as Env-pseudotyped viruses showed a wide range of virus infectivity. This variation in infectivity correlated with plasma viral load but not with protein expression levels in transfected cells, suggesting a more functional env gene may lead to the higher viral load in HIV-1 infected individuals.
The Example that follows also describes that neutralization susceptibility to antibodies in autologous and heterologous plasma samples was determined for multiple Envs (3-6) from each of 15 individuals. While reduced neutralization was observed for autologous viruses, heterologous neutralization was detected and could be divided into two distinct groups: plasma with broadly cross-reactive neutralization (N=9) and plasma that was poor at cross-reactive neutralization (N=6). Plasmas with stronger neutralizing activity against heterologous viruses more potently neutralized contemporaneous autologous viruses. Analysis of Env sequences in plasma from both groups revealed a three amino acid signature in the V4 region, proximal to the co-receptor binding site, that was associated with greater neutralization potency and breadth.
The present invention thus relates to HIV-1 subtype C Envs that retain the signature associated with potent antibody responses (e.g., signature sites 393, 397, and 413) and methods of using same as vaccine immunogens. The invention further relates to such Envs for use as diagnostic targets in diagnostic tests. The invention further relates to the use of wildtype (WT) virus sequences in the preparation of a polyvalent HIV-1 vaccine (U.S. Provisional Application No. 61/282,526, filed Feb. 25, 2010). Sequences that can be included in such a polyvalent vaccine for B cell response include env and for T helper and cytotoxic T cell response include gag, pol, nef and tat sequences (U.S. application Ser. No. 11/990, 222, filed Aug. 23, 2006).
The vaccine antigens (immunogens) of the invention (e.g. Envs sequences that retain the signature associated with potent antibody responses) can be chemically synthesized and purified using methods well known in the art. The immunogens can also be synthesized by well-known recombinant DNA techniques. Nucleic acids encoding the immunogens of the invention can be used as components of, for example, a DNA vaccine wherein the encoding sequence is administered as naked DNA or, for example, a minigene encoding the immunogen can be present in a viral vector. The encoding sequence can be present, for example, in a replicating or non-replicating adenoviral vector, an adeno-associated virus vector, an attenuated mycobacterium tuberculosis vector, a Bacillus Calmette Guerin (BCG) vector, a vaccinia or Modified Vaccinia Ankara (MVA) vector, another pox virus vector, recombinant polio and other enteric yin's vector, Salmonella species bacterial vector, Shigella species bacterial vector, Venezuelean Equine Encephalitis Virus (VEE) vector, a Semliki Forest Virus vector, or a Tobacco Mosaic Virus vector. The encoding sequence, can also be expressed as a DNA plasmid with, for example, an active promoter such as a CMV promoter. Other live vectors can also be used to express the sequences of the invention. Expression of the immunogen of the invention can be induced in a patient's own cells, by introduction into those cells of nucleic acids that encode the immunogen, preferably, using codons and promoters that optimize expression in human cells. Examples of methods of making and using DNA vaccines are disclosed in, for example, U.S. Pat. Nos. 5,580,859, 5,589,466, and 5,703,055.
The invention includes compositions comprising an immunologically effective amount of the immunogen of the invention (e.g., gp160 or gp140) or fragment thereof (e.g., gp41, gp120, either alone or associated with lipids, or fragments of gp120), or nucleic acid sequence encoding same, in a pharmaceutically acceptable delivery system. The compositions can be used for prevention and/or treatment of immunodeficiency virus infection (e.g., in a human). The compositions of the invention can be formulated using adjuvants (e.g., alum, AS021 (from GSK), oligo CpGs, MF59 or Emulsigen), emulsifiers, pharmaceutically-acceptable carriers or other ingredients routinely provided in vaccine compositions. Optimum formulations can be readily designed by one of ordinary skill in the art and can include formulations for immediate release and/or for sustained release, and for induction of systemic immunity and/or induction of localized mucosal immunity (e.g, the formulation can be designed for intranasal administration). The present compositions can be administered by any convenient route including subcutaneous, intranasal, intrarectal, intravaginal, oral, intramuscular, or other parenteral or enteral route, or combinations thereof. The immunogens can be administered in an amount sufficient to induce an immune response, e.g., as a single dose or multiple doses. Optimum immunization schedules can be readily determined by the ordinarily skilled artisan and can vary with the patient, the composition and the effect sought.
Examples of compositions and administration regimens of the invention include consensus or mosaic gag genes and consensus or mosaic nef genes and consensus or mosaic poi genes and consensus Env with an Env that retains the above-described signature or mosaic Env with an Env that retains the above-described signature, expressed as, for example, a DNA prime recombinant Vesicular stomatitis virus boost and a recombinant Env protein boost for antibody, a poxvirus prime such as NYVAC and a protein Env oligomer boost, or fragment thereof, or DNA prime recombinant adenovirus boost and Env protein boost, or, for just antibody induction, only the recombinant envelope gp120 or gp140 as a protein in an adjuvant. (See U.S. application Ser. No. 10/572,638, PCT/US2006/032907, U.S. application Ser. Nos. 11/990,222 and 12/192,015.)
The invention contemplates the direct use of both the immunogen of the invention and/or nucleic acid encoding same and/or the immunogen expressed as a minigene in the vectors indicated above. For example, a minigene encoding the immunogen can be used as a prime and/or boost,
It will be appreciated from a reading of this disclosure that the whole Envelope gene can be used or portions thereof (i.e., as minigenes). In the case of expressed proteins, protein subunits can be used.
As pointed out above, the invention also relates to diagnostic targets and diagnostic tests. For example, a signature-retaining Env of the invention can be expressed by transient or stable transfection of mammalian cells (or they can be expressed, for example, as recombinant Vaccinia virus proteins). The protein can be used in ELISA, Luminex bead test, or other diagnostic tests to detect antibodies to the transmitted/founder virus in a biological sample from a patient at the earliest stage of HIV infection.
The present invention also relates to antibodies specific for signature-retaining Envs of the invention, and fragments of such antibodies, and to methods of using same to inhibit infection of cells of a subject by HIV-1. The method comprises administering to the subject (e.g., a human subject) the HIV-1 specific antibody, or fragment thereof, in an amount and under conditions such that the antibody, or fragment thereof, inhibits infection.
In accordance with the invention, the antibodies can be administered prior to contact of the subject or the subject's immune system/cells with HIV-1 or after infection of vulnerable cells. Administration prior to contact or shortly thereafter can maximize inhibition of infection of vulnerable cells of the subject (e.g., T-cells).
As indicated above, either the intact antibody or fragment (e.g., antigen binding fragment) thereof can be used in the method of the present invention. Exemplary functional fragments (regions) include scFv, Fv, Fab′, Fab and F(ab′)2 fragments. Single chain antibodies can also be used. Techniques for preparing suitable fragments and single chain antibodies are well known in the art. (See, for example, U.S. Pat. Nos. 5,855,866; 5,877,289; 5,965,132; 6,093,399; 6,261,535; 6,004,555; 7,417,125 and 7,078,491 and WO 98/45331.)
The antibodies, and fragments thereof, described above can be formulated as a composition (e.g., a pharmaceutical composition). Suitable compositions can comprise the antibody (or antibody fragment) dissolved or dispersed in a pharmaceutically acceptable carrier (e.g., an aqueous medium). The compositions can be sterile and can in an injectable form. The antibodies (and fragments thereof) can also be formulated as a composition appropriate for topical administration to the skin or mucosa. Such compositions can take the form of liquids, ointments, creams, gels, pastes or aerosols. Standard formulation techniques can be used in preparing suitable compositions. The antibodies can be formulated so as to be administered as a post-coital douche or with a condom.
The antibodies and antibody fragments of the invention show their utility for prophylaxis in, for example, the following settings:
i) in the setting of anticipated known exposure to HIV-1 infection, the antibodies described herein (or binding fragments thereof) can be administered prophylactically (e.g., IV or topically) as a microbiocide,
ii) in the setting of known or suspected exposure, such as occurs in the setting of rape victims, or commercial sex workers, or in any sexual transmission with out condom protection, the antibodies described herein (or fragments thereof) can be administered as post-exposure prophylaxis, e.g., IV or topically, and
iii) in the setting of Acute HIV infection (AHI), antibodies described herein (or binding fragments thereof) can be administered as a treatment for AHI to control the initial viral load and preserve the CD4+T cell pool and prevent CD4+T cell destruction.
Suitable dose ranges can depend, for example, on the antibody and on the nature of the formulation and route of administration. Optimum doses can be determined by one skilled in the art without undue experimentation. Doses of antibodies in the range of 10 ng to 20 μg/ml can be suitable.
The present invention also includes nucleic acid sequences encoding the antibodies, or fragments thereof, described herein. The nucleic acid sequences can be present in an expression vector operably linked to a promoter. The invention further relates to isolated cells comprising such a vector and to a method of making the antibodies, or fragments thereof, comprising culturing such cells under conditions such that the nucleic acid sequence is expressed and the antibody, or fragment, is produced.
Certain aspects of the invention can be described in greater detail in the non-limiting Example that follows. (U.S. Application Ser. Nos. 61/322,725, 61/322,821, and 61/322,663 are incorporated herein by reference.)
Elicitation of broad and potent neutralizing antibodies to HIV-1 has been challenging. However, recent studies indicate that neutralizing antibodies are required for an AIDS vaccine to effectively control HIV-1 infection. To understand the ability of HIV-1 to induce neutralizing antibodies during natural infection, comprehensive autologous and heterologous neutralization assays were performed using multiple Env-pseudoviruses from each subtype C infected individual, and identified a three amino acid signature in the V4 region, proximal to the co-receptor binding site, that was associated with greater neutralization potency and breadth. Identification of a signature for eliciting broadly reactive neutralizing antibody responses has important implications for the development of vaccine candidates capable of inducing neutralizing antibodies to HIV-1. These results also showed the presence of autologous neutralization in the contemporaneous plasmas with stronger neutralizing activity against heterologous viruses, clonal expansion of viruses in the majority of the chronically infected individuals, and a positive correlation between the infectivity of Env-pseudoviruses and viral loads in the infected individuals.
Amplification of HIV-1 env Genes.
Plasma samples were collected from 37 HIV-1 positive individuals enrolled in a study of contemporary HIV-1 strains in Ndola, Zambia. The study was approved by the ethics committee of the Tropical Disease Research Centre, the Duke University Institutional Review Board, and the National Institutes of Health. Viral RNA was extracted from the plasma and reverse transcribed into cDNA using Superscript III (Invitrogen; Carlsbad, Calif.). Multiple rev/env genes from each individual were obtained by using single genome amplification (SGA), followed by the addition of a CMV promoter to the 5′ end of the SGA products using pPCR technology as previously described [38].
Single Round Infection Assay.
pPCR products were cotransfected with an env-deficient HIV-1 backbone plasmid (pSG3Δenv) into 293T cells in a 24 well plate using FuGENE6 transfection reagent (Roche Diagnostics; Indianapolis, Ind.). Briefly, pPCR DNA (150 ng) and pSG3Δenv DNA (150 ng) were mixed with 1.2 μl of FuGENE6 (FuGENE:DNA ratio at 3 μl:1 μg) in a total volume of 20 it with serum free DMEM, incubated for 30 minutes and added to 293T cells (70% confluence) seeded one day earlier at 5×104 per well. Forty-eight hours after transfection, supernatants were harvested. Equal volumes of pseudovirions were added to TZM-bl cells with DEAE (5 μg/ml) in a 96 well plate (200 μl). Cultures were incubated for 48 hrs at 37° C. with 5% CO2. Supernatants (100 μl) from infected TZM-bl cells were removed and 100 μl of Bright-Glo Luciferase Assay substrate with buffer (Promega; Madison, Wis.) was added to the cells. Following a 2-minute incubation, 100 μl of cell lysates were added to a solid black 96 well plate. Luminescence was measured with a Wallac 1420 Multilabel Counter (PerkinElmer: Waltham, Mass.). The TCID50 was determined as described previously [39].
Neutralization Assay.
HIV-1 neutralization was measured as a reduction in luciferase activity after a single round infection of TZM-bl cells as previously described [39,40]. Equal amounts of pseudovirions (200 TCID50) were used in each reaction. Neutralization titers against pPCR pseudovirions were determined for 16 plasma samples (15 autologous and 1 heterologous to the tested Env pseudoviruses) and one HIV-1 positive serum control. An amphotropic murine leukemia Env pseudovirus was also included as non-specific neutralization control.
Western Blot.
Forty-eight hours after cotransfection with pPCR products and pSG3Δenv, 293T cells were lysed with 250 μl of lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 20 mM EDTA, 1% Triton-X100, 0.1% SDS, pH 7.4). Cell lysates were mixed with 6× denaturing sample buffer (300 mM Tris-HCl pH 6.8, 12 mM EDTA, 12% SDS, 864 mM 2-mercaptoethanol, 60% glycerol, 0.05% bromophenol blue). Samples were boiled for 10 minutes and then loaded on a NuPAGE Novex 4-12% Bis-Tris ge (Invitrogen; Carlesbad, Calif.). After the samples were transferred to a nitrocellulose membrane, the membrane was blocked in PBS containing 1% casein and 0.01% NaN3 for 1 hr. The blot was reacted with an HIV-1 positive serum (1:500) and a mouse mAb 13D5 (1 μg/ml) to the HIV-1 Env protein. Finally, the membrane was reacted with an IRDye800 conjugated affinity purified goat anti-human IgG antibody (Rockland Immunochemicals; Gilbertsville, Pa.) and an Alexa-Fluor 680 conjugated goat anti-mouse IgG antibody (Invitrogen; Carlesbad, Calif.). Fluorescence was detected and the density of Env protein and p24 bands was determined with an Odyssey Infrared Imaging system (LiCor Biosciences; Lincoln, Nebr.). The level of Env protein in the transfected cells was expressed as fold differences over the amount of p24 Gag antigen in the same cells.
Sequence Analysis.
Sequences of SGA env amplicons were obtained by cycle-sequencing and dye terminator methods with an ABI 3730×1 genetic analyzer (Applied Biosystems; Foster City, Calif.). Individual sequence fragments for each env SGA were assembled and edited using the Sequencher program 4.7 (Gene Codes; Ann Arbor, Mich.). The env sequences were aligned using CLUSTAL W [41], and Genecutter (www.hiv.lanl.gov) for obtaining codon aligned files that could be used for signature analysis. The phylogenetic trees were constructed with either Neighbor joining or Maximum likelihood methods, as specified in the legends.
Hierarchical Clustering Analysis.
The neutralizing potential of plasma (the log reciprocal dilution ID50 scores) was organized into patterns of similar neutralizing potency when tested against the panel of envelopes used in this study using the Los Alamos National Lab web-based heatmap interface (http://www.hiv.lanl.gov/content/sequence/HEATMAP/heatmap.html) and the statistical package R [42] The test panel of envelopes was resampled 10,000 times using a random-with-replacement bootstrap strategy, and this identified two robust and distinctive clusters recurring in 96% of the bootstrap samples: one group with little cross-neutralizing potential and another group with the ability to cross-neutralize multiple heterologous C clade viruses [43]. These two groups were used for subsequent signature analysis. The same strategy was used to identify statistically robust clusters of the Envs with like-neutralizing susceptibility. Clusters with high bootstrap values typically only included a few Envs each, and these Env clusters were typically found within a single subject. Therefore, we decided to organize Env pseudoviruses by individual in heatmap figure, while retaining the information regarding significant bootstrap clusters, shown superimposed onto the figure.
Signature Analysis.
We first divided the envelope sequences into the two groups, based on heatmap clustering patterns, that indicated whether they were derived from an individual with plasma that had a cross-reactive neutralizing profile or a weakly neutralizing profile. Alignments used for signature analysis were generated with GeneCutter (www.hiv.lanl.gov) to provide codon-aligned DNA for phylogenetic analysis. Phylogenetically corrected methods were used to identify all signature sites. Phylogenetic correction is critical because observed patterns in data can result either from correlations imposed by the initial historical emergence of a lineage of viruses (founder effects), or in the case of HIV-1, be a consequence of recent biological interactions. Not accounting for founder effects can lead to erroneous statistical conclusions [44]. The sequence of the virus depends on its full evolutionary history, while causal correlations are manifest in correlations with recent changes. The separation of the two effects, i.e. a phylogenetic correction, enables one to estimate the impact recent changes on phenotype. This requires statistical reconstruction the genealogical relationships between the viruses and an estimate of ancestral states of the viruses. We implemented this through maximum likelihood phylogenies, and tested for phenotypic associations with mutational patterns based on change or stasis in a given amino acid, when compared to the most recent common ancestor, adapting the phylogenetically corrected Fisher's exact methods first developed in Bhattacharya et al [44]. We screened the mutational pattern of each amino acid found every column in the alignment. To correct for multiple tests we used a q-value to assess the false discovery rate [45]. The four strongest associations found each had had a p-value of 0.03, and q-value of 0.27, thus is it is like that at least one of the signatures identified is a false positive, and only borderline significance was obtained overall, hence these signatures should be considered in a hypothesis-raising framework, as potentially interesting sites that merit further investigation.
Nucleotide Sequences Accession Numbers.
GenBank accession numbers off all env gene sequences are GU329048-GU329523.
Genetic Analysis of Full-Length env Genes.
Plasma samples were collected from 50 CHI individuals in Ndola, Zambia in 2005. A total of 474 complete env genes were obtained from 37 of 50 subjects by SGA. Negative PCR results for the remaining 13 subjects were due to either low or undetectable plasma viral loads. An average of thirteen env SGAs (range 5-23) were sequenced from each of the 37 PCR-positive subjects (Table 1). Phylogenetic analysis showed that a majority of subjects (n=34) were infected with HIV-1 subtype C (
Identical or closely related env sequences were identified in 13 of 37 (35%) of subjects (Table 2), suggesting clonal expansion of minority viral populations in the HIV-1 infected individuals. In these subjects, clonally expanded Envs comprised 9-38% of the sampled env population. Sequences in each population of clonally expanded envelopes formed a tight cluster with zero or limited branch lengths in the phylogenetic trees (
Functional Analysis of the env Quasispecies in Infected Individuals.
A pPCR method that adds a CMV promoter to SGA amplicons [38] was used for high throughput functional screening of multiple env genes from each infected individual. Env-pseudotyped viruses were generated by cotransfecting 293T cells with pPCR products and an Env-defective backbone plasmid (SG3Δenv). Infectivity was measured by luciferase activity in TZM-bl cells. Out of a total of 474 Envs examined, 377 (80%) were found to be functional (Table 1). All newly characterized env genes were functional in eight subjects, whereas in a few subjects only a small fraction of any genes were functional (10% and 18% for ZM419 and ZM393, respectively). In three individuals (ZM373, ZM380 and ZM382), all Env-pseudotyped viruses were highly infectious and differed in infectivity by only a few folds (
Recombinant genes were found in 11 env sequences from 6 individuals. Examination of their functionality showed that 8 (73%) were biologically functional while the other three were not (
Given the range of variability in Env function, it was of interest to determine whether the level of Env infectivity in pseudovirus assays was associated with either protein expression levels or with plasma viral RNA load. As shown in
These results suggested that variation in infectivity was not determined by expression levels, protein sizes or the cleavage efficiency of gp160 in transfected cells. It is possible that the amount of Envs that were sufficient for infection in transfected cells was above the threshold required for the production of fully infectious pseudoviruses since only a small number of Env spikes are present on mature HIV-1 particles [47,48]. Non-infectious pseudoviruses were associated with either the absence of Env expression or with the expression of truncated Envs; these cases were explained by either premature stop codons or frame-shifting deletions by sequence analysis.
Autologous and Heterologus Neutralization.
A checkerboard-style dataset of the neutralizing activity in autologous and heterologous plasma samples was generated by assaying plasma samples against multiple representative Env-pseudotyped viruses from each subject. Because of the limited supply of plasma, it was only possible to perform neutralization assays with 60 Env-pseudotyped viruses from 15 subjects (14 subtype C and one A/C recombinant) using plasma samples from these 15 subjects plus one additional plasma sample (ZM383). Multiple env genes (range 3-6) from each subject were selected to represent the viral population as seen in a Maximum-likelihood tree analysis (
Neutralization results are summarized in
All Env-pseudotyped viruses were then assayed for neutralization susceptibility by heterologous plasma. Plasma samples were segregated into low (L) and high (H) neutralization potency groups by heatmap analysis (
Neutralization Signature Analysis.
The identification of plasma samples with potent and cross-reactive Nabs prompted us to ask if there were signature sequences present among the Envs from those subjects that associated with the potent neutralizing activity. Because each plasma sample represented one value per individual, we used only the consensus sequence of all variants within an individual for the signature study, thereby capturing the most common amino in each position found in each subject. We scanned each position in the alignment using the Maximum likelihood tree correction method previously described [44] to look for amino acids associated with either strong or weak subtype C cross-reactive neutralization responses, based on the two distinctive clusters shown in the heatmap (
Four signature sites were identified: 3 in gp120 and one in gp41. Each site had a p-value of 0.03, but a q-value of 0.27; thus it may be that one or more of the signatures identified were false positives (
Interestingly, autologous neutralizing activity was significantly higher (p=0.0016) in the H plasma group than in the L plasma group of heterologous neutralization potencies (
In summary, we characterized multiple HIV-1 env genes in each of 37 individuals using SGA and pPCR methods, Sequence and functional analysis of the env gene quasispecies in each individual showed multiple lineages of viruses, frequent clonal expansions, a trend towards a positive correlation between viral loads and the infectivity of Env pseudoviruses, highly variable infectivity among env gene quasispecies populations, and signature amino acids in the V4 region of sequences in plasma with high levels of neutralization activity. Identification of such neutralization signatures may have implications for development of effective HIV-1 vaccines.
As expected for chronic HIV-1 infection, a highly divergent viral quasispecies population was detected in the subject studies here. Intra-subject env gene diversity was as high as 8% in this study. Using our recently established pPCR method, we were able to characterize the phenotypic properties of a large number of env genes from each HIV-1 infected individual by generating pseudoviruses without cloning SGA PCR products. Analysis of the infectivity of 474 Env pseudoviruses from 37 individuals revealed that a high percentage (80%) of the env genes were functional. This is consistent to our previous report with a smaller number of env genes [38] but much higher than the reported rate (10%) from env gene populations characterized by bulk PCR from PBMC [25]. The different rates were likely due to differences in methods and sample sources used to obtain env genes. Functional analysis showed that the infectivity of Env pseudoviruses from the same individuals varied significantly (1-2 logs) in most individuals. This is similar to plasma derived env genes from one individual [51], but different from PBMC derived env genes which showed uniform infectivity from the same individual [25]. Examination of Env proteins in the transfected cells showed that the variation of infectivity among Env pseudoviruses in each individual was not correlated with in vitro expression levels. With an intact env open reading frame, the non-functionality of the env genes may be caused by mutations that affect either incorporation into virus particles or poor interaction with CD4 and coreceptors on target cells. Interestingly, a positive correlation trend was observed between the levels of infectivity of Env-pseudoviruses and viral loads in the donor subjects, suggesting that viruses with more infectious env genes may lead to a high level of viral loads in vivo.
Sequence analysis showed that 13 of 37 individuals (35%) carried one or two clusters of closely related sequences among the quasispecies viral populations. These sequences, unique to each individual and conserved within that individual, were identical or nearly identical to each other, Since each sequence was obtained from independent PCR by SGA, they represent in vivo derived independent viral genomes. The env sequences related to but beginning to diverge from the identical or nearly identical sequences, as well as recombinants between these conserved sets and more diverse viruses from that individual, indicate that there is clonal expansion of some viruses in the infected individuals. Overall, they accounted for 9-38% of viral population. Clonal expansion of some virus species was found in different tissues in HIV-1 infected individuals [52]. It is not clear what the sources are for clonally expanded viruses in plasma. It is possible that clonally expanded viruses have an advantage due to replication fitness or immune escape mutations. Since clonal expansion viruses contain amino acids that differ from those in viruses in the major viral population, and because they can recombine with other viruses (
An analysis of checkerboard neutralization data with multiple representative env genes from each individual using autologous and heterologous plasma samples revealed a signature of three amino acids that associated with potent neutralizing plasmas. Glycines associated with signature sites 393 and 397 in the V4 loop of gp120 could impact antibody neutralization in several ways. Firstly, the loss of electrostatic charge associated with side chains can perturb antibody binding by modulating the electrostatic potential of the cognate epitope. Secondly, due to their lack of side chains, glycines exhibit higher flexibility that might affect conformational motions in the V4 loop. Flexibility and less charge repulsion due to glycines at positions 393 and 397 might allow V4 to sample a wide variety of conformations. In contrast, the signature 413N might play a role by mediating N-linked glycosylation and potentially inhibiting V4 loop movement by steric hindrence.
Site 413 is proximal to the 17b and CCRS binding sites, and is adjacent to the proximal binding motif RIKQ (HXB2 residues 419-422). A recent glycopeptide mass spectroscopy (MS) study found that glycans were indeed attached at this site in the group M consensus HIV-1 envelope, CON-S gp140 [53]. Both high-resolution methods, online high performance liquid chromatography-electrospray ionization mass spectrometry (HPLC/ESI-MS) and offline HPLC followed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) determined that the potential glycosylation site 413 is in fact glycosylated. It is, however, not clear how a glycosylation at this position is associated with induction of higher levels of neutralizing antibody titers in vivo. This position is quite variable; indeed the glycosylation site could be enriched in Envs associated with potent neutralization activity because it is selected against by a strong response rather than being required to elicit a strong response.
In a recent study, a neutralization signature was investigated in subtype C sera using 36 sera against a panel of 5 subtype B and 5 C subtype viruses [20]. No clear clustering of serological patterns was found to enable tracking of the signature patterns. However, a correlation between the V1-V4 loop length and the cross-subtype neutralizing potential was observed. In the current study, we did not find evidence for such a correlation when comparing the total V1-V4 lengths or the lengths of V1, V2, and V4 separately by comparing the of lengths of the consensus Env from each individual grouped by the high and low neutralization plasma using a Wilcoxon rank statistic. It could have simply because that the number of the samples was underpowered to show this effect. The other possibility is that all samples in the current study were from CHI individuals in whom the length of variable loops had already become longer during the evolution of immune evasion.
Previous studies also showed that the alpha-2 helix was under strong selection pressure [50] and this region was found associated with resistance to autologous neutralization of subtype C viruses [49], The V4 region is immediately downstream from the alpha-2 helix and they are structurally close to each other. It was also found that the C3V4 region is likely responsible for inducing autologous and heterologous Nabs [18]. The newly identified three signature amino acids were located in the vicinity of the previously reported region targeted by Nabs, suggesting that tertiary structure may play an important role in the induction of Nabs against subtype C viruses. Identification of this potential signature may help to design a new generation Env immunogens that elicit potent and broadly reactive Nabs, at least for subtype C which is the most prevalent HIV-1 subtype in the world.
All documents and other information sources cited herein are hereby incorporated in their entirety by reference.
This application claims priority from U.S. Prov. Appln. No. 61/332,262, filed May 7, 2010, the entire content of which is incorporated herein by reference.
This invention was made with government support under Grant Nos. A1067854, A135351, and A164518 awarded by the National Institutes of Health. The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 61332262 | May 2010 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/US2011/000810 | May 2011 | US |
| Child | 13671490 | US |
