A single mutation turns a non-binding germline-like predecessor of broadly neutralizing antibody into a binding antibody to HIV-1 envelope glycoproteins

Broadly neutralizing antibodies (bnAbs) against human immunodeficiency virus (HIV)-1 are rare in natural infection and elicitation of HIV-1 bnAbs has not been achieved by any vaccine candidates. We and others have reported that HIV-1 bnAbs are highly diversified from their germline-like predecessors, and the germline-like predecessors of bnAbs lack measurable binding to HIV-1 envelope (Env) glycoproteins, suggesting that Env structures containing the epitopes of bnAbs may not initiate somatic maturation pathway, which may partially explain the rarity of HIV-1 bnAbs. To determine the minimum mutations required for converting non-binding germline-like predecessors to Env-binding antibodies, we started with the bnAb b12 as a prototype and generated six “chimeric” scFv b12 variants by sequentially replacing the heavy chain V-segment (HV), D(J)-segment [HD(J)] in the heavy chain variable region (VH), and the whole light chain variable region (VL) in b12 germline-like predecessor with the mature counterparts. We tested the recombinant scFv variants for binding and neutralizing activities. Results showed that a single point mutation in germline D-segment was enough to convert nonbinding germline-like b12 to an Env-binding antibody. Replacement with either mature HV or mature VL also made the germline-like b12 bind to Env, but none of single segment replacements conferred neutralization ability to the germline antibody. Mature VL in combination with mature HD(J), or mature HV, or both conferred increasing neutralization activity to the germline antibody. However, hybrid scFv, mature VH / germline VL did not neutralize the virus, suggesting the importance of mature VL in neutralizing the virus. These results may have implications for vaccine development.     

Characterization of germline antibody libraries from human umbilical cord blood and selection of monoclonal antibodies to viral envelope glycoproteins: Implications for mechanisms of immune evasion and design of vaccine immunogens

We have previously observed that all known HIV-1 broadly neutralizing antibodies (bnAbs) are highly divergent from germline antibodies in contrast to bnAbs against Hendra virus, Nipah virus and SARS coronavirus (SARS CoV). We have hypothesized that because the germline antibodies are so different from the mature HIV-1-specific bnAbs they may not bind the epitopes of the mature antibodies and provided the first evidence to support this hypothesis by using individual putative germline-like predecessor antibodies. To further validate the hypothesis and understand initial immune responses to different viruses, two phage-displayed human cord blood-derived IgM libraries were constructed which contained mostly germline antibodies or antibodies with very low level of somatic hypermutations. They were panned against different HIV-1 envelope glycoproteins (Envs), SARS CoV protein receptor-binding domain (RBD), and soluble Hendra virus G protein (sG). Despite a high sequence and combinatorial diversity observed in the cord blood-derived IgM antibody repertoire, no enrichment for binders of Envs was observed in contrast to considerable specific enrichments produced with panning against RBD and sG; one of the selected monoclonal antibodies (against the RBD) was of high (nM) affinity with only few somatic mutations. These results further support and expand our initial hypothesis for fundamental differences in immune responses leading to elicitation of bnAbs against HIV-1 compared to SARS CoV and Hendra virus. HIV-1 uses a strategy to minimize or eliminate strong binding of germline antibodies to its Env; in contrast, SARS CoV and Hendra virus, and perhaps other viruses causing acute infections, can bind germline antibody or minimally somatically mutated antibodies with relatively high affinity which could be one of the reasons for the success of sG and RBD as vaccine immunogens.     

Identification of Non-HIV Immunogens That Bind to Germline b12 Predecessors and Prime for Elicitation of Cross-clade Neutralizing HIV-1 Antibodies

A fundamental challenge for developing an effective and safe HIV-1 vaccine is to identify vaccine immunogens that can initiate and maintain immune responses leading to elicitation of broadly neutralizing HIV-1 antibodies (bnAbs) through complex maturation pathways. We have previously found that HIV-1 envelope glycoproteins (Env) lack measurable binding to putative germline predecessors of known bnAbs and proposed to search for non-HIV immunogens that could initiate their somatic maturation. Using bnAb b12 as a model bnAb and yeast display technology, we isolated five (poly)peptides from plant leaves, insects, E. coli strains, and sea water microbes that bind to b12 putative germline and intermediate antibodies. Rabbit immunization with the (poly)peptides alone induced high titers of cross-reactive antibodies that neutralized HIV-1 isolates SF162 and JRFL. Priming rabbits with the (poly)peptides followed by boosts with trimeric gp140_SF162 and then resurfaced Env (RSC3) induced antibodies that competed with mature b12 and neutralized tier 1 and 2 viruses from clade B, C and E, while control rabbits without (poly)peptide priming induced antibodies that did not compete with mature b12 and neutralized fewer isolates. The degree of competition with mature b12 for binding to gp140_SF162 correlated with the neutralizing activity of the rabbit IgG. Reversing the order of the two boosting immunogens significantly affected the binding profile and neutralization potency of the rabbit IgG. Our study is the first to provide evidence that appears to support the concept that non-HIV immunogens may initiate immune responses leading to elicitation of cross-clade neutralizing antibodies.     

Recombinant HIV Envelope Proteins Fail to Engage Germline Versions of Anti-CD4bs bNAbs

Vaccine candidates for HIV-1 so far have not been able to elicit broadly neutralizing antibodies (bNAbs) although they express the epitopes recognized by bNAbs to the HIV envelope glycoprotein (Env). To understand whether and how Env immunogens interact with the predicted germline versions of known bNAbs, we screened a large panel (N:56) of recombinant Envs (from clades A, B and C) for binding to the germline predecessors of the broadly neutralizing anti-CD4 binding site antibodies b12, NIH45-46 and 3BNC60. Although the mature antibodies reacted with diverse Envs, the corresponding germline antibodies did not display Env-reactivity. Experiments conducted with engineered chimeric antibodies combining the mature and germline heavy and light chains, respectively and vice-versa, revealed that both antibody chains are important for the known cross-reactivity of these antibodies. Our results also indicate that in order for b12 to display its broad cross-reactivity, multiple somatic mutations within its VH region are required. A consequence of the failure of the germline b12 to bind recombinant soluble Env is that Env-induced B-cell activation through the germline b12 BCR does not take place. Our study provides a new explanation for the difficulties in eliciting bNAbs with recombinant soluble Env immunogens. Our study also highlights the need for intense efforts to identify rare naturally occurring or engineered Envs that may engage the germline BCR versions of bNAbs.     

Affinity maturation and characterization of a human monoclonal antibody against HIV-1 gp41

The human D5 monoclonal antibody binds to the highly conserved hydrophobic pocket on the N-terminal heptad repeat (NHR) trimer of HIV-1 gp41 and exhibits modest yet relatively broad neutralization activity. Both binding and neutralization depend on residues in the complementarity determining regions (CDRs) of the D5 IgG variable domains on heavy chain (VH) and light chain (VL). In an effort to increase neutralization activity to a wider range of HIV-1 strains, we have affinity matured the parental D5 scFv by randomizing selected residues in 5 of its 6 CDRs. The resulting scFv variants derived from four different CDR changes showed enhanced binding affinities to gp41 NHR mimetic (5-helix) which correlated to improved neutralization potencies by up to 8-fold. However, when converted to IgG1s, these D5 variants had up to a 12-fold reduction in neutralization potency over their corresponding scFvs despite their slightly enhanced in vitro binding affinities. Remarkably, D5 variant IgG1s bearing residue changes in CDRs that interact with epitope residues N-terminal to the hydrophobic pocket (such as VH CDR3 and VL CDR3) retained more neutralization potency than those containing residue changes in pocket-interacting CDRs (such as VH CDR2). These results provide compelling evidence for the existence of a steric block to an IgG that extends to the gp41 NHR hydrophobic pocket region, and can be a useful guide for developing therapeutic antibodies and vaccines circumventing this block.     

Germline-like predecessors of broadly neutralizing antibodies lack measurable binding to HIV-1 envelope glycoproteins: Implications for evasion of immune responses and design of vaccine immunogens

Several human monoclonal antibodies (hmAbs) including b12, 2G12, and 2F5 exhibit relatively potent and broad HIV-1-neutralizing activity. However, their elicitation in vivo by vaccine immunogens based on the HIV-1 envelope glycoprotein (Env) has not been successful. We have hypothesized that HIV-1 has evolved a strategy to reduce or eliminate the immunogenicity of the highly conserved epitopes of such antibodies by using “holes” (absence or very weak binding to these epitopes of germline antibodies that is not sufficient to initiate and/or maintain an efficient immune response) in the human germline B cell receptor (BCR) repertoire. To begin to test this hypothesis we have designed germline-like antibodies corresponding most closely to b12, 2G12, and 2F5 as well as to X5, m44, and m46 which are cross-reactive but with relatively modest neutralizing activity as natively occurring antibodies due to size and/or other effects. The germline-like X5, m44, and m46 bound with relatively high affinity to all tested Envs. In contrast, germline-like b12, 2G12, and 2F5 lacked measurable binding to Envs in an ELISA assay although the corresponding mature antibodies did. These results provide initial evidence that Env structures containing conserved vulnerable epitopes may not initiate humoral responses by binding to germline antibodies. Even if such responses are initiated by very weak binding undetectable in our assay it is likely that they will be outcompeted by responses to structures containing the epitopes of X5, m44, m46, and other antibodies that bind germline BCRs with much higher affinity/avidity. This hypothesis, if further supported by data, could contribute to our understanding of how HIV-1 evades immune responses and offer new concepts for design of effective vaccine immunogens.     

HIV-1 immunogens and strategies to drive antibody responses towards neutralization breadth

Despite enormous efforts no HIV-1 vaccine has been developed that elicits broadly neutralizing antibodies (bNAbs) to protect against infection to date. The high antigenic diversity and dense N-linked glycan armor, which covers nearly the entire HIV-1 envelope protein (Env), are major roadblocks for the development of bNAbs by vaccination. In addition, the naive human antibody repertoire features a low frequency of exceptionally long heavy chain complementary determining regions (CDRH3s), which is a typical characteristic that many HIV-1 bNAbs use to penetrate the glycan armor. Native-like Env trimer immunogens can induce potent but strain-specific neutralizing antibody responses in animal models but how to overcome the many obstacles towards the development of bNAbs remains a challenge. Here, we review recent HIV-1 Env immunization studies and discuss strategies to guide strain-specific antibody responses towards neutralization breadth.     

Affinity maturation and characterization of a human monoclonal antibody against HIV-1 gp41

The human D5 monoclonal antibody binds to the highly conserved hydrophobic pocket on the N-terminal heptad repeat (NHR) trimer of HIV-1 gp41 and exhibits modest yet relatively broad neutralization activity. Both binding and neutralization depend on residues in the complementarity determining regions (CDRs) of the D5 IgG variable domains on heavy chain (VH) and light chain (VL). In an effort to increase neutralization activity to a wider range of HIV-1 strains, we have affinity matured the parental D5 scFv by randomizing selected residues in 5 of its 6 CDRs. The resulting scFv variants derived from four different CDR changes showed enhanced binding affinities to gp41 NHR mimetic (5-helix) which correlated to improved neutralizationpotencies by up to 8-fold. However, when converted to IgG1s, these D5 variants had up to a 12-fold reduction in neutralization potency over their corresponding scFvs despite their slightly enhanced in vitro binding affinities. Remarkably, D5 variant IgG1s bearing residue changes in CDRs that interact with epitope residues N-terminal to the hydrophobic pocket (such as VH CDR3 and VL CDR3) retained more neutralization potency than those containing residue changes in pocket-interacting CDRs (such as CDR2). These results provide compelling evidence for the existence of a steric block to an IgG that extends to the gp41 NHR hydrophobic pocket region, and can be a useful guide for developing therapeutic antibodies and vaccines circumventing this block.     

基于核糖体展示技术进行抗狂犬病毒人源抗体的制备

构建了核糖体展示人源抗狂犬病毒单链抗体(scFv)库,筛选制备特异抗狂犬病毒糖蛋白(RVGp)的稳定性人源抗体.应用核糖体抗体库技术,从经狂犬病毒Vero疫苗免疫的志愿者外周血淋巴细胞中分离、构建核糖体展示scFv基因库.体外转录翻译后,以RVGp重组蛋白作筛选抗原,采用亲和富集法淘选RVGp特异性scFv抗体基因.在原核系统pET22b(+)/BL21(DE3)中实现scFv抗体片段的可溶性表达,ELISA鉴定阳性克隆.然后对筛选的scFv进行稳定性改构,构建VH-Lc-VK稳定性抗体,并对其生物学活性进行初步研究.成功构建了库容量约为6.2×1012的核糖体展示scFv抗体基因库.在180个筛选克隆中,克隆RB24、RB71、RB109和RB156显示出较高的ELISA值,其基因序列分析结果显示,它们是全新的人源抗RVGp抗体.改构后的抗RVGp VH-Lc-VK抗体的稳定性明显改进,可特异识别RVGp并有效中和狂犬病毒,抑制狂犬病毒对靶细胞的感染.以上结果表明,人源抗RVGp特异性抗体的获得,为狂犬病的有效预防、诊断和治疗提供了新的途径,而且将为其他人源抗体的制备提供理论依据和技术基础.     

Cross-neutralization of SARS-CoV-2 by HIV-1 specific broadly neutralizing antibodies and polyclonal plasma

Cross-reactive epitopes (CREs) are similar epitopes on viruses that are recognized or neutralized by same antibodies. The S protein of SARS-CoV-2, similar to type I fusion proteins of viruses such as HIV-1 envelope (Env) and influenza hemagglutinin, is heavily glycosylated. Viral Env glycans, though host derived, are distinctly processed and thereby recognized or accommodated during antibody responses. In recent years, highly potent and/or broadly neutralizing human monoclonal antibodies (bnAbs) that are generated in chronic HIV-1 infections have been defined. These bnAbs exhibit atypical features such as extensive somatic hypermutations, long complementary determining region (CDR) lengths, tyrosine sulfation and presence of insertions/deletions, enabling them to effectively neutralize diverse HIV-1 viruses despite extensive variations within the core epitopes they recognize. As some of the HIV-1 bnAbs have evolved to recognize the dense viral glycans and cross-reactive epitopes (CREs), we assessed if these bnAbs cross-react with SARS-CoV-2. Several HIV-1 bnAbs showed cross-reactivity with SARS-CoV-2 while one HIV-1 CD4 binding site bnAb, N6, neutralized SARS-CoV-2. Furthermore, neutralizing plasma antibodies of chronically HIV-1 infected children showed cross neutralizing activity against SARS-CoV-2 pseudoviruses. Collectively, our observations suggest that human monoclonal antibodies tolerating extensive epitope variability can be leveraged to neutralize pathogens with related antigenic profile.     

Recombinant single-chain Fv antibodies that recognize the p25 protein of the Maedi-Visna virus

Single chain Fv (scFv) antibodies (generated by phage display technology, molecules representing new and efficient tools in the research and diagnostics of infectious diseases) against the capsid protein (p25) of Maedi-Visna virus were selected. Several clones of p25 specific scFv antibodies were identified; one of them was expressed as a soluble scFv molecule, purified by immobilized metal-affinity chromatography and further characterized by sequencing and determination of the kinetic equilibrium association constant. Sequence analysis showed that the rearranged VL and VH domains of the analyzed scFv clone used sequences from the VL3 family (germline DPL16/VL3.1) and VH1 family (germline VH20), respectively. The kinetic equilibrium association constant was determined as KA = 1.12 +/- 0.52 L/mumol.     

Gene cloning, bacterial expression, in vitro refolding, and characterization of a single-chain Fv antibody against PreS1(21-47) fragment of HBsAg

The murine monoclonal antibody 125E11 is an IgG which recognizes PreS1(21-47) fragment of large hepatitis B surface antigen. It has been successfully used for clinical detection of HBV virion in serum of hepatitis B patients. In present study, the genes of variable region in heavy chain (VH) and light chain (VL) of 125E11 have been cloned. Sequence analysis of cloned VH gene and VL gene showed that they had general characterization of immunoglobin variable region genes. According to Kabat classification, VH gene and VL gene belong to VH10 family, subgroup IIID and Vkappa family subgroup I, respectively. An expression vector of 125E11 single-chain Fv antibody fusion protein, in which VH and VL peptide were connected by a flexible linker (Gly(4)Ser)(3), was constructed. The scFv fusion protein was highly expressed in Escherichia coli mainly in inclusion body form. Using urea and pH gradient gel filtration method, the refolding of scFv was efficiently achieved. The refolding efficiency reached about 11% and 2.7 mg refolded scFv was obtained from 1L of culture. The binding activity and specificity of 125E11 scFv against PreS1(21-47)-containing antigen were also analyzed.     

Elicitation of potent serum neutralizing antibody responses in rabbits by immunization with an HIV-1 clade C trimeric Env derived from an Indian elite neutralizer

Evaluating the structure-function relationship of viral envelope (Env) evolution and the development of broadly cross-neutralizing antibodies (bnAbs) in natural infection can inform rational immunogen design. In the present study, we examined the magnitude and specificity of autologous neutralizing antibodies induced in rabbits by a novel HIV-1 clade C Env protein (1PGE-THIVC) vis-à-vis those developed in an elite neutralizer from whom the env sequence was obtained that was used to prepare the soluble Env protein. The novel 1PGE-THIVC Env trimer displayed a native like pre-fusion closed conformation in solution as determined by small angle X-ray scattering (SAXS) and negative stain electron microscopy (EM). This closed spike conformation of 1PGE-THIVC Env trimers was correlated with weak or undetectable binding of non-neutralizing monoclonal antibodies (mAbs) compared to neutralizing mAbs. Furthermore, 1PGE-THIVC SOSIP induced potent neutralizing antibodies in rabbits to autologous virus variants. The autologous neutralizing antibody specificity induced in rabbits by 1PGE-THIVC was mapped to the C3/V4 region (T362/P401) of viral Env. This observation agreed with electron microscopy polyclonal epitope mapping (EMPEM) of the Env trimer complexed with IgG Fab prepared from the immunized rabbit sera. Our study demonstrated neutralization of sequence matched and unmatched autologous viruses by serum antibodies induced in rabbits by 1PGE-THIVC and also highlighted a comparable specificity for the 1PGE-THIVC SOSIP trimer with that seen with polyclonal antibodies elicited in the elite neutralizer by negative-stain electron microscopy polyclonal epitope (ns-EMPEM) mapping.     

HIV-1 Receptor Binding Site-Directed Antibodies Using a VH1-2 Gene Segment Orthologue Are Activated by Env Trimer Immunization

Broadly neutralizing antibodies (bNAbs) isolated from chronically HIV-1 infected individuals reveal important information regarding how antibodies target conserved determinants of the envelope glycoprotein (Env) spike such as the primary receptor CD4 binding site (CD4bs). Many CD4bs-directed bNAbs use the same heavy (H) chain variable (V) gene segment, VH1-2*02, suggesting that activation of B cells expressing this allele is linked to the generation of this type of Ab. Here, we identify the rhesus macaque VH1.23 gene segment to be the closest macaque orthologue to the human VH1-2 gene segment, with 92% homology to VH1-2*02. Of the three amino acids in the VH1-2*02 gene segment that define a motif for VRC01-like antibodies (W50, N58, flanking the HCDR2 region, and R71), the two identified macaque VH1.23 alleles described here encode two. We demonstrate that immunization with soluble Env trimers induced CD4bs-specific VH1.23-using Abs with restricted neutralization breadth. Through alanine scanning and structural studies of one such monoclonal Ab (MAb), GE356, we demonstrate that all three HCDRs are involved in neutralization. This contrasts to the highly potent CD4bs-directed VRC01 class of bNAb, which bind Env predominantly through the HCDR2. Also unlike VRC01, GE356 was minimally modified by somatic hypermutation, its light (L) chain CDRs were of average lengths and it displayed a binding footprint proximal to the trimer axis. These results illustrate that the Env trimer immunogen used here activates B cells encoding a VH1-2 gene segment orthologue, but that the resulting Abs interact distinctly differently with the HIV-1 Env spike compared to VRC01.     

Bacterial single-chain antibody fragments, specific for carcinoembryonic antigen.

We have produced single-chain antibody (scFv) fragments in bacteria specific for carcinoembryonic antigen (CEA). Polymerase chain reaction (PCR) was used for the cloning and modification of the heavy and light variable regions (VH and VL) of the mouse monoclonal antibody (MAb) CB-CEA.1. A 14-amino acid linker was used in the synthesis of the scFv gene. The VH and VL regions were amplified from cDNA by PCR using 5' end FR1 and 3' end constant region primers, and then sequenced. VH was then amplified by PCR using an exact 5' end FR1 primer, and a phosphorylated (PP) 3' end primer for J2 that also encoded the first 7 amino acids of the linker. VL was amplified with a PP 5' end primer for FR1, also encoding the remaining 7 amino acids of the linker, and a 3' end primer for J5, plus a stop codon and a BglII restriction site. The fragments were ligated and reamplified with the PP VH 5' and VL 3' end primers. The VH-linker-VL structure was blunt-cloned into expression vectors bearing the tryptophan promoter and pelB or ompA signal peptide sequences. Culture supernatant, bacteria pellet and periplasm preparations were assayed in Western blot and a protein of about 27 kDa was identified with rabbit antibodies specific for the Fab of CB-CEA.1. Bacterial supernatant and periplasm preparations also inhibited the recognition of CEA by HRP-labeled CB-CEA.1 in enzyme-linked immunosorbent assay (ELISA). Periplasm preparations were purified by affinity chromatography with specific anti-idiotypic MAbs. The Western blot of the eluates identified a protein of approximately 27 kDa that blocked the recognition of CEA by HRP-labeled CB-CEA.1 in ELISA. The VH-linker-VL structure was cloned into a vector bearing the lacZ promoter and the pelB signal peptide. The recombinant bacterial clones also expressed about 27 kDa scFv, specific for CEA.     

Antigenic analysis of the HIV-1 envelope trimer implies small differences between structural states 1 and 2

The conformationally dynamic HIV-1 envelope trimer (Env) is the target of broadly neutralizing antibodies (bnAbs) that block viral entry. Single-molecule Förster resonance energy transfer (smFRET) has revealed that HIV-1 Env exists in at least three conformational states on the virion. Prior to complete host–receptor engagement (State 3), Env resides most prevalently in the smFRET-defined State 1, which is preferentially recognized by most bnAbs that are elicited by natural infection. smFRET has also revealed that soluble trimers containing prefusion-stabilizing disulfide and isoleucine-to-proline substitutions reside primarily in State 2, which is a required intermediate between States 1 and 3. While high-resolution Env structures have been determined for States 2 and 3, the structure of these trimers in State 1 is unknown. To provide insight into the State 1 structure, here we characterized antigenic differences between smFRET-defined states and then correlated these differences with known structural differences between States 2 and 3. We found that cell surface–expressed Env was enriched in each state using state-enriching antibody fragments or small-molecule virus entry inhibitors and then assessed binding to HIV-1 bnAbs preferentially binding different states. We observed small but consistent differences in binding between Env enriched in States 1 and 2, and a more than 10-fold difference in binding to Env enriched in these states versus Env enriched in State 3. We conclude that structural differences between HIV-1 Env States 1 and 3 are likely more than 10-fold greater than those between States 1 and 2, providing important insight into State 1.     

Optimal Combinations of Broadly Neutralizing Antibodies for Prevention and Treatment of HIV-1 Clade C Infection

The identification of a new generation of potent broadly neutralizing HIV-1 antibodies (bnAbs) has generated substantial interest in their potential use for the prevention and/or treatment of HIV-1 infection. While combinations of bnAbs targeting distinct epitopes on the viral envelope (Env) will likely be required to overcome the extraordinary diversity of HIV-1, a key outstanding question is which bnAbs, and how many, will be needed to achieve optimal clinical benefit. We assessed the neutralizing activity of 15 bnAbs targeting four distinct epitopes of Env, including the CD4-binding site (CD4bs), the V1/V2-glycan region, the V3-glycan region, and the gp41 membrane proximal external region (MPER), against a panel of 200 acute/early clade C HIV-1 Env pseudoviruses. A mathematical model was developed that predicted neutralization by a subset of experimentally evaluated bnAb combinations with high accuracy. Using this model, we performed a comprehensive and systematic comparison of the predicted neutralizing activity of over 1,600 possible double, triple, and quadruple bnAb combinations. The most promising bnAb combinations were identified based not only on breadth and potency of neutralization, but also other relevant measures, such as the extent of complete neutralization and instantaneous inhibitory potential (IIP). By this set of criteria, triple and quadruple combinations of bnAbs were identified that were significantly more effective than the best double combinations, and further improved the probability of having multiple bnAbs simultaneously active against a given virus, a requirement that may be critical for countering escape in vivo. These results provide a rationale for advancing bnAb combinations with the best in vitro predictors of success into clinical trials for both the prevention and treatment of HIV-1 infection.     

Pharmacokinetics and Immunogenicity of Broadly Neutralizing HIV Monoclonal Antibodies in Macaques

The identification of highly potent broadly neutralizing antibodies (bnAbs) against HIV-1, and success in preventing SHIV infection following their passive administration, have increased the likelihood that immunotherapeutic strategies can be adopted to prevent and treat HIV-1 infection. However, while broad and potent neutralizing activity is an essential prerequisite, in vivo properties such as good circulatory stability and non-immunogenicity are equally critical for developing a human treatment. In the present study, glycoforms of the bnAbs 10-1074, NIH45-46^G54W, 10E8, PGT121, PGT128, PGT145, PGT135, PG9, PG16, VRC01 and b12 were produced by Agrobacterium-mediated transient transfection of Nicotiana benthamiana and assessed following administration in rhesus macaques. The results indicate that (i) N-glycans within the VL domain impair plasma stability of plant-derived bnAbs and (ii) while PGT121 and b12 exhibit no immunogenicity in rhesus macaques after multiple injections, VRC01, 10-1074 and NIH45-46^G54W elicit high titer anti-idiotypic antibodies following a second injection. These anti-idiotypic antibodies specifically bind the administered bnAb or a close family member, and inhibit the bnAb in neutralization assays. These findings suggest that specific mutations in certain bnAbs contribute to their immunogenicity and call attention to the prospect that these mutated bnAbs will be immunogenic in humans, potentially compromising their value for prophylaxis and therapy of HIV-1.     

Unique C2V3 Sequence in HIV-1 Envelope Obtained from Broadly Neutralizing Plasma of a Slow Progressing Patient Conferred Enhanced Virus Neutralization

Broadly neutralizing antibodies to HIV-1 usually develops in chronic infections. Here, we examined the basis of enhanced sensitivity of an env clone amplified from cross neutralizing plasma of an antiretroviral naïve chronically infected Indian patient (ID50 >600-fold higher compared to other autologous env clones). The enhanced autologous neutralization of pseudotyped viruses expressing the sensitive envelope (Env) was associated with increased sensitivity to reagents and monoclonal antibodies targeting distinct sites in Env. Chimeric viruses constructed by swapping fragments of sensitive Env into resistant Env backbone revealed that the presence of unique residues within C2V3 region of gp120 governed increased neutralization. The enhanced virus neutralization was also associated with low CD4 dependence as well as increased binding of Env trimers to IgG1b12 and CD4-IgG2 and was independent of gp120 shedding. Our data highlighted vulnerabilities in the Env obtained from cross neutralizing plasma associated with the exposure of discontinuous neutralizing epitopes and enhanced autologous neutralization. Such information may aid in Env-based vaccine immunogen design.     

Affinity maturation to improve human monoclonal antibody neutralization potency and breadth against hepatitis C virus

A potent neutralizing antibody to a conserved hepatitis C virus (HCV) epitope might overcome its extreme variability, allowing immunotherapy. The human monoclonal antibody HC-1 recognizes a conformational epitope on the HCV E2 glycoprotein. Previous studies showed that HC-1 neutralizes most HCV genotypes but has modest potency. To improve neutralization, we affinity-matured HC-1 by constructing a library of yeast-displayed HC-1 single chain Fv (scFv) mutants, using for selection an E2 antigen from one of the poorly neutralized HCVpp. We developed an approach by parallel mutagenesis of the heavy chain variable (VH) and κ-chain variable (Vk) genes separately, then combining the optimized VH and Vk mutants. This resulted in the generation of HC-1-related scFv variants exhibiting improved affinities. The best scFv variant had a 92-fold improved affinity. After conversion to IgG1, some of the antibodies exhibited a 30-fold improvement in neutralization activity. Both surface plasmon resonance and solution kinetic exclusion analysis showed that the increase in affinity was largely due to a lowering of the dissociation rate constant, Koff. Neutralization against a panel of HCV pseudoparticles and infectious 2a HCV virus improved with the affinity-matured IgG1 antibodies. Interestingly, some of these antibodies neutralized a viral isolate that was not neutralized by wild-type HC-1. Moreover, propagating 2a HCVcc under the selective pressure of WT HC-1 or affinity-matured HC-1 antibodies yielded no viral escape mutants and, with the affinity-matured IgG1, needed 100-fold less antibody to achieve complete virus elimination. Taken together, these findings suggest that affinity-matured HC-1 antibodies are excellent candidates for therapeutic development.