Requirements for the Induction of Broadly Neutralizing Antibodies against HIV-1 by Vaccination

A study of the induction of broadly neutralizing antibodies (bNAbs) in HIV-infected patients and vaccinated subjects revealed the main criteria for the formation of bNAbs (the duration of exposure to a viral antigen, the effect of the diversity of HIV variants, and the removal of barriers associated with the Env-dependent defense mechanisms of HIV-1). Modified trimers of the HIV-1 envelope protein (Env) exposed on virus-like particles (VLP) have unique properties: they (i) modulate the exposure of binding sites (bs) of the CD4 receptor and co-receptor; (ii) create steric restrictions for contact with bNAbs; (iii) increase the Env presentation density, thus enhancing the immune response; (iv) form stable trimers that do not induce off-target immune responses; and (v) allow additional modifications to their structure and construction of a platform with immunostimulating molecules. Immunization using a heterologous subtype-cross prime–boost regime with modified trimeric Env is capable of inducing somatic hypermutation levels necessary for the formation of bNAbs.     

HIV-1中和抗体和基于抗体的疫苗设计

研制一种安全有效并能广泛使用的HIV疫苗对于预防和控制HIV的流行具有重要的意义。尽管人类在HIV-1病原学和免疫学方面的认识不断取得新的进步,对于HIV-1而言,普遍认为诱导保护性中和抗体的科学障碍很难逾越。在抗击HIV-1的感染中传统的疫苗策略不能提供保护。然而,近来的研究揭示在小部分HIV-1感染病人的血清中存在的某些抗体能够中和大多数的HIV-1毒株,对这些血清抗体的深入分析有助于人们揭示抗体识别的病毒表位,这些研究表明自然产生的能够中和HIV-1的中和抗体的发现可能引导未来疫苗设计的思路。高分辨率的结构信息将揭示Env 和中和抗体(Nab)结合区原子水平的结构,这些信息能够帮助设计更好的免疫原。     

Anti-V3 Monoclonal Antibodies Display Broad Neutralizing Activities against Multiple HIV-1 Subtypes

Background

The V3 loop of the HIV-1 envelope (Env) glycoprotein gp120 was identified as the “principal neutralizing domain” of HIV-1, but has been considered too variable to serve as a neutralizing antibody (Ab) target. Structural and immunochemical data suggest, however, that V3 contains conserved elements which explain its role in binding to virus co-receptors despite its sequence variability. Despite this evidence of V3 conservation, the ability of anti-V3 Abs to neutralize a significant proportion of HIV-1 isolates from different subtypes (clades) has remained controversial.

Methods

HIV-1 neutralization experiments were conducted in two independent laboratories to test human anti-V3 monoclonal Abs (mAbs) against pseudoviruses (psVs) expressing Envs of diverse HIV-1 subtypes from subjects with acute and chronic infections. Neutralization was defined by 50% inhibitory concentrations (IC₅₀), and was statistically assessed based on the area under the neutralization titration curves (AUC).

Results

Using AUC analyses, statistically significant neutralization was observed by ≥1 anti-V3 mAbs against 56/98 (57%) psVs expressing Envs of diverse subtypes, including subtypes A, AG, B, C and D. Even when the 10 Tier 1 psVs tested were excluded from the analysis, significant neutralization was detected by ≥1 anti-V3 mAbs against 46/88 (52%) psVs from diverse HIV-1 subtypes. Furthermore, 9/24 (37.5%) Tier 2 viruses from the clade B and C standard reference panels were neutralized by ≥1 anti-V3 mAbs. Each anti-V3 mAb tested was able to neutralize 28–42% of the psVs tested. By IC₅₀ criteria, 40/98 (41%) psVs were neutralized by ≥1 anti-V3 mAbs.

Conclusions

Using standard and new statistical methods of data analysis, 6/7 anti-V3 human mAbs displayed cross-clade neutralizing activity and revealed that a significant proportion of viruses can be neutralized by anti-V3 Abs. The new statistical method for analysis of neutralization data provides many advantages to previously used analyses.     

Immunological Fingerprints of Controllers Developing Neutralizing HIV-1 Antibodies

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Conformational Plasticity in the HIV-1 Fusion Peptide Facilitates Recognition by Broadly Neutralizing Antibodies

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Robust Neutralizing Antibodies Elicited by HIV-1 JRFL Envelope Glycoprotein Trimers in Nonhuman Primates

Host cell-mediated proteolytic cleavage of the human immunodeficiency virus type 1 (HIV-1) gp160 precursor glycoprotein into gp120 and gp41 subunits is required to generate fusion-competent envelope glycoprotein (Env) spikes. The gp120-directed broadly neutralizing monoclonal antibodies (bNabs) isolated from HIV-infected individuals efficiently recognize fully cleaved JRFL Env spikes; however, nonneutralizing gp120-directed monoclonal antibodies isolated from infected or vaccinated subjects recognize only uncleaved JRFL spikes. Therefore, as an immunogen, cleaved spikes that selectively present desired neutralizing epitopes to B cells may elicit cross-reactive neutralizing antibodies. Accordingly, we inoculated nonhuman primates (NHPs) with plasmid DNA encoding transmembrane-anchored, cleaved JRFL Env or by electroporation (EP). Priming with DNA expressing soluble, uncleaved gp140 trimers was included as a comparative experimental group of NHPs. DNA inoculation was followed by boosts with soluble JRFL gp140 trimers, and control NHPs were inoculated with soluble JRFL protein trimers without DNA priming. In the TZM-bl assay, elicitation of neutralizing antibodies against HIV-1 tier 1 isolates was robust following the protein boost. Neutralization of tier 2 isolates was detected, but only in animals primed with plasmid DNA and boosted with trimeric protein. Using the more sensitive A3R5 assay, consistent neutralization of both clade B and C tier 2 isolates was detected from all regimens assessed in the current study, exceeding levels achieved by our previous vaccine regimens in primates. Together, these data suggest a potential advantage of B cell priming followed by a rest interval and protein boosting to present JRFL Env spikes to the immune system to better generate HIV-1 cross-clade neutralizing antibodies.     

A Fusion Intermediate gp41 Immunogen Elicits Neutralizing Antibodies to HIV-1

The membrane-proximal external region (MPER) of the human immunodeficiency virus, type 1 (HIV-1) envelope glycoprotein subunit gp41 is targeted by potent broadly neutralizing antibodies 2F5, 4E10, and 10E8. These antibodies recognize linear epitopes and have been suggested to target the fusion intermediate conformation of gp41 that bridges viral and cellular membranes. Anti-MPER antibodies exert different degrees of membrane interaction, which is considered to be the limiting factor for the generation of such antibodies by immunization. Here we characterize a fusion intermediate conformation of gp41 (gp41_int-Cys) and show that it folds into an elongated ∼12-nm-long extended structure based on small angle x-ray scattering data. Gp41_int-Cys was covalently linked to liposomes via its C-terminal cysteine and used as immunogen. The gp41_int-Cys proteoliposomes were administered alone or in prime-boost regimen with trimeric envelope gp140_CA018 in guinea pigs and elicited high anti-gp41 IgG titers. The sera interacted with a peptide spanning the MPER region, demonstrated competition with broadly neutralizing antibodies 2F5 and 4E10, and exerted modest lipid binding, indicating the presence of MPER-specific antibodies. Although the neutralization potency generated solely by gp140_CA018 was higher than that induced by gp41_int-Cys, the majority of animals immunized with gp41_int-Cys proteoliposomes induced modest breadth and potency in neutralizing tier 1 pseudoviruses and replication-competent simian/human immunodeficiency viruses in the TZM-bl assay as well as responses against tier 2 HIV-1 in the A3R5 neutralization assay. Our data thus demonstrate that liposomal gp41 MPER formulation can induce neutralization activity, and the strategy serves to improve breadth and potency of such antibodies by improved vaccination protocols.     

Minimally Mutated HIV-1 Broadly Neutralizing Antibodies to Guide Reductionist Vaccine Design

An optimal HIV vaccine should induce broadly neutralizing antibodies (bnAbs) that neutralize diverse viral strains and subtypes. However, potent bnAbs develop in only a small fraction of HIV-infected individuals, all contain rare features such as extensive mutation, insertions, deletions, and/or long complementarity-determining regions, and some are polyreactive, casting doubt on whether bnAbs to HIV can be reliably induced by vaccination. We engineered two potent VRC01-class bnAbs that minimized rare features. According to a quantitative features frequency analysis, the set of features for one of these minimally mutated bnAbs compared favorably with all 68 HIV bnAbs analyzed and was similar to antibodies elicited by common vaccines. This same minimally mutated bnAb lacked polyreactivity in four different assays. We then divided the minimal mutations into spatial clusters and dissected the epitope components interacting with those clusters, by mutational and crystallographic analyses coupled with neutralization assays. Finally, by synthesizing available data, we developed a working-concept boosting strategy to select the mutation clusters in a logical order following a germline-targeting prime. We have thus developed potent HIV bnAbs that may be more tractable vaccine goals compared to existing bnAbs, and we have proposed a strategy to elicit them. This reductionist approach to vaccine design, guided by antibody and antigen structure, could be applied to design candidate vaccines for other HIV bnAbs or protective Abs against other pathogens.     

Characterization of a Large Panel of Rabbit Monoclonal Antibodies against HIV-1 gp120 and Isolation of Novel Neutralizing Antibodies against the V3 Loop

We recently reported the induction of potent, cross-clade neutralizing antibodies (nAbs) against Human Immunodeficiency Virus type-1 (HIV-1) in rabbits using gp120 based on an M-group consensus sequence. To better characterize these antibodies, 93 hybridomas were generated, which represent the largest panel of monoclonal antibodies (mAbs) ever generated from a vaccinated rabbit. The single most frequently recognized epitope of the isolated mAbs was at the very C-terminal end of the protein (APTKAKRRVVEREKR), followed by the V3 loop. A total of seven anti-V3 loop mAbs were isolated, two of which (10A3 and 10A37) exhibited neutralizing activity. In contrast to 10A3 and most other anti-V3 loop nAbs, 10A37 was atypical with its epitope positioned more towards the C-terminal half of the loop. To our knowledge, 10A37 is the most potent and broadly neutralizing anti-V3 loop mAb induced by vaccination. Interestingly, all seven anti-V3 loop mAbs competed with PGT121, suggesting a possibility that early induction of potent anti-V3 loop antibodies could prevent induction of more broadly neutralizing PGT121-like antibodies that target the conserved base of the V3 loop stem.     

An Efficiently Cleaved HIV-1 Clade C Env Selectively Binds to Neutralizing Antibodies

An ideal HIV-1 Env immunogen is expected to mimic the native trimeric conformation for inducing broadly neutralizing antibody responses. The native conformation is dependent on efficient cleavage of HIV-1 Env. The clade B isolate, JRFL Env is efficiently cleaved when expressed on the cell surface. Here, for the first time, we report the identification of a native clade C Env, 4-2.J41 that is naturally and efficiently cleaved on the cell surface as confirmed by its biochemical and antigenic characteristics. In addition to binding to several conformation-dependent neutralizing antibodies, 4-2.J41 Env binds efficiently to the cleavage-dependent antibody PGT151; thus validating its native cleaved conformation. In contrast, 4-2.J41 Env occludes non-neutralizing epitopes. The cytoplasmic-tail of 4-2.J41 Env plays an important role in maintaining its conformation. Furthermore, codon optimization of 4-2.J41 Env sequence significantly increases its expression while retaining its native conformation. Since clade C of HIV-1 is the prevalent subtype, identification and characterization of this efficiently cleaved Env would provide a platform for rational immunogen design.     

Early Low-Titer Neutralizing Antibodies Impede HIV-1 Replication and Select for Virus Escape

Single genome sequencing of early HIV-1 genomes provides a sensitive, dynamic assessment of virus evolution and insight into the earliest anti-viral immune responses in vivo. By using this approach, together with deep sequencing, site-directed mutagenesis, antibody adsorptions and virus-entry assays, we found evidence in three subjects of neutralizing antibody (Nab) responses as early as 2 weeks post-seroconversion, with Nab titers as low as 1∶20 to 1∶50 (IC₅₀) selecting for virus escape. In each of the subjects, Nabs targeted different regions of the HIV-1 envelope (Env) in a strain-specific, conformationally sensitive manner. In subject CH40, virus escape was first mediated by mutations in the V1 region of the Env, followed by V3. HIV-1 specific monoclonal antibodies from this subject mapped to an immunodominant region at the base of V3 and exhibited neutralizing patterns indistinguishable from polyclonal antibody responses, indicating V1–V3 interactions within the Env trimer. In subject CH77, escape mutations mapped to the V2 region of Env, several of which selected for alterations of glycosylation. And in subject CH58, escape mutations mapped to the Env outer domain. In all three subjects, initial Nab recognition was followed by sequential rounds of virus escape and Nab elicitation, with Nab escape variants exhibiting variable costs to replication fitness. Although delayed in comparison with autologous CD8 T-cell responses, our findings show that Nabs appear earlier in HIV-1 infection than previously recognized, target diverse sites on HIV-1 Env, and impede virus replication at surprisingly low titers. The unexpected in vivo sensitivity of early transmitted/founder virus to Nabs raises the possibility that similarly low concentrations of vaccine-induced Nabs could impair virus acquisition in natural HIV-1 transmission, where the risk of infection is low and the number of viruses responsible for transmission and productive clinical infection is typically one.     

VSV-Displayed HIV-1 Envelope Identifies Broadly Neutralizing Antibodies Class-Switched to IgG and IgA

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Structural and Biochemical Characterization of Cysteinylation in Broadly Neutralizing Antibodies to HIV-1

Antibodies with exceptional breadth and potency have been elicited in some individuals during natural HIV-1 infection. Elicitation and affinity maturation of broadly neutralizing antibodies (bnAbs) is therefore the central goal of HIV-1 vaccine development. The functional properties of bnAbs also make them attractive as immunotherapeutic agents, which has led to their production and optimization for passive immunotherapy. This process requires in vitro manufacturing and monitoring of any heterogeneous expression, especially when subpopulations of antibodies are produced with varying levels of biological activity. Post-translational modification (PTM) of antibodies can contribute to heterogeneity and is the focus of this study. Specifically, we have investigated cysteinylation in a bnAb lineage (PCDN family) targeting the N332-glycan supersite on the surface envelope glycoprotein (Env) of HIV-1. This PTM is defined by capping of unpaired cysteine residues with molecular cysteine. Through chromatography and mass spectrometry, we were able to characterize subpopulations of cysteinylated and non-cysteinylated antibodies when expressed in mammalian cells. The crystal structures of two PCDN antibodies represent the first structures of a cysteinylated antibody and reveal that the cysteinylation in this case is located in CDRH3. Biophysical studies indicate that cysteinylation of these HIV-1 antibodies does not interfere with antigen binding, which has been reported to occur in other cysteinylated antibodies. As such, these studies highlight the need for further investigation of cysteinylation in anti-HIV and other bnAbs.     

Epitope Mapping of Broadly Neutralizing HIV-2 Human Monoclonal Antibodies

Recent studies have shown that natural infection by HIV-2 leads to the elicitation of high titers of broadly neutralizing antibodies (NAbs) against primary HIV-2 strains (T. I. de Silva, et al., J. Virol. 86:930–946, 2012; R. Kong, et al., J. Virol. 86:947–960, 2012; G. Ozkaya Sahin, et al., J. Virol. 86:961–971, 2012). Here, we describe the envelope (Env) binding and neutralization properties of 15 anti-HIV-2 human monoclonal antibodies (MAbs), 14 of which were newly generated from 9 chronically infected subjects. All 15 MAbs bound specifically to HIV-2 gp120 monomers and neutralized heterologous primary virus strains HIV-2_7312A and HIV-2_ST. Ten of 15 MAbs neutralized a third heterologous primary virus strain, HIV-2_UC1. The median 50% inhibitory concentrations (IC₅₀s) for these MAbs were surprisingly low, ranging from 0.007 to 0.028 μg/ml. Competitive Env binding studies revealed three MAb competition groups: CG-I, CG-II, and CG-III. Using peptide scanning, site-directed mutagenesis, chimeric Env constructions, and single-cycle virus neutralization assays, we mapped the epitope of CG-I antibodies to a linear region in variable loop 3 (V3), the epitope of CG-II antibodies to a conformational region centered on the carboxy terminus of V4, and the epitope(s) of CG-III antibodies to conformational regions associated with CD4- and coreceptor-binding sites. HIV-2 Env is thus highly immunogenic in vivo and elicits antibodies having diverse epitope specificities, high potency, and wide breadth. In contrast to the HIV-1 Env trimer, which is generally well shielded from antibody binding and neutralization, HIV-2 is surprisingly vulnerable to broadly reactive NAbs. The availability of 15 human MAbs targeting diverse HIV-2 Env epitopes can facilitate comparative studies of HIV/SIV Env structure, function, antigenicity, and immunogenicity.