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1.  Molecular Evolution of Broadly Neutralizing Llama Antibodies to the CD4-Binding Site of HIV-1 
PLoS Pathogens  2014;10(12):e1004552.
To date, no immunization of humans or animals has elicited broadly neutralizing sera able to prevent HIV-1 transmission; however, elicitation of broad and potent heavy chain only antibodies (HCAb) has previously been reported in llamas. In this study, the anti-HIV immune responses in immunized llamas were studied via deep sequencing analysis using broadly neutralizing monoclonal HCAbs as a guides. Distinct neutralizing antibody lineages were identified in each animal, including two defined by novel antibodies (as variable regions called VHH) identified by robotic screening of over 6000 clones. The combined application of five VHH against viruses from clades A, B, C and CRF_AG resulted in neutralization as potent as any of the VHH individually and a predicted 100% coverage with a median IC50 of 0.17 µg/ml for the panel of 60 viruses tested. Molecular analysis of the VHH repertoires of two sets of immunized animals showed that each neutralizing lineage was only observed following immunization, demonstrating that they were elicited de novo. Our results show that immunization can induce potent and broadly neutralizing antibodies in llamas with features similar to human antibodies and provide a framework to analyze the effectiveness of immunization protocols.
Author Summary
Developing a vaccine against HIV-1 is a priority, but it remains unclear whether immunizations in humans can elicit potent broadly neutralizing antibodies able to prevent HIV-1 transmission. Llamas possess heavy chain only antibodies and conventional heavy and light chain antibodies. We previously reported the heavy chain only antibody J3, which potently neutralizes more than 95% of HIV strains, and was induced by immunization. Here we immunized two further llamas and elicited three novel broadly neutralizing heavy chain only antibodies, which were identified by high-throughput screening. These neutralizing llama antibodies target different areas of the CD4-binding site of the virus, therefore breadth and potency are increased when they are used in combination. To gain greater understanding of how the llama immunizations worked, deep sequencing of the HIV binding region of the antibodies was performed. This revealed that the antibodies were matured fully only in response to the protein immunogens. Furthermore, the VHH elicited in different animals, while sharing functional hallmarks, were encoded by distinct sequences and thus could not have been identified by a deep sequencing analysis alone. Our results show that immunization can potentially induce protective antibodies in llamas and provide a method to more extensively evaluate immunization studies.
doi:10.1371/journal.ppat.1004552
PMCID: PMC4270772  PMID: 25522326
2.  Neutralisation of HIV-1 cell-cell spread by human and llama antibodies 
Retrovirology  2014;11(1):83.
Background
Direct cell-cell spread of HIV-1 is a very efficient mode of viral dissemination, with increasing evidence suggesting that it may pose a considerable challenge to controlling viral replication in vivo. Much current vaccine research involves the study of broadly neutralising antibodies (bNabs) that arise during natural infection with the aims of eliciting such antibodies by vaccination or incorporating them into novel therapeutics. However, whether cell-cell spread of HIV-1 can be effectively targeted by bNabs remains unclear, and there is much interest in identifying antibodies capable of efficiently neutralising virus transmitted by cell-cell contact.
Results
In this study we have tested a panel of bNAbs for inhibition of cell-cell spread, including some not previously evaluated for inhibition of this mode of HIV-1 transmission. We found that three CD4 binding site antibodies, one from an immunised llama (J3) and two isolated from HIV-1-positive patients (VRC01 and HJ16) neutralised cell-cell spread between T cells, while antibodies specific for glycan moieties (2G12, PG9, PG16) and the MPER (2F5) displayed variable efficacy. Notably, while J3 displayed a high level of potency during cell-cell spread we found that the small size of the llama heavy chain-only variable region (VHH) J3 is not required for efficient neutralisation since recombinant J3 containing a full-length human heavy chain Fc domain was significantly more potent. J3 and J3-Fc also neutralised cell-cell spread of HIV-1 from primary macrophages to CD4+ T cells.
Conclusions
In conclusion, while bNabs display variable efficacy at preventing cell-cell spread of HIV-1, we find that some CD4 binding site antibodies can inhibit this mode of HIV-1 dissemination and identify the recently described llama antibody J3 as a particularly potent inhibitor. Effective neutralisation of cell-cell spread between physiologically relevant cell types by J3 and J3-Fc supports the development of VHH J3 nanobodies for therapeutic or prophylactic applications.
doi:10.1186/s12977-014-0083-y
PMCID: PMC4189184
HIV-1; Antibody; Virological synapse; Cell-cell; Neutralisation; CD4; VHH
4.  In Tribute to Joep Lange 
Retrovirology  2014;11(1):82.
doi:10.1186/s12977-014-0082-z
PMCID: PMC4169638
5.  TRANSMISSIBLE DOG CANCER GENOME REVEALS THE ORIGIN AND HISTORY OF AN ANCIENT CELL LINEAGE 
Science (New York, N.Y.)  2014;343(6169):437-440.
Canine transmissible venereal tumor (CTVT) is the oldest known somatic cell lineage. It is a transmissible cancer that propagates naturally in dogs. We sequenced the genomes of two CTVT tumors and found that CTVT has acquired 1.9 million somatic substitution mutations and bears evidence of exposure to ultraviolet light. CTVT is remarkably stable and lacks subclonal heterogeneity despite thousands of rearrangements, copy number changes and retrotransposon insertions. More than 10,000 genes carry non-synonymous variants and 646 genes have been lost. CTVT first arose in a dog with low genomic heterozygosity that may have lived approximately 11,000 years ago. The cancer spawned by this individual dispersed across continents approximately 500 years ago. Our results provide a genetic identikit of an ancient dog and demonstrate the robustness of mammalian somatic cells to survive for millennia despite a massive mutation burden.
doi:10.1126/science.1247167
PMCID: PMC3918581  PMID: 24458646
6.  On the concept and elucidation of endogenous retroviruses 
Endogenous retrovirus (ERV) genomes integrated into the chromosomal DNA of the host were first detected in chickens and mice as Mendelian determinants of Gag and Env proteins and of the release of infectious virus particles. The presence of ERV was confirmed by DNA hybridization. With complete host genomes available for analysis, we can now see the great extent of viral invasion into the genomes of numerous vertebrate species, including humans. ERVs are found at many loci in host DNA and also in the genomes of large DNA viruses, such as herpesviruses and poxviruses. The evolution of xenotropism and cross-species infection is discussed in the light of the dynamic relationship between exogenous and endogenous retroviruses.
doi:10.1098/rstb.2012.0494
PMCID: PMC3758183  PMID: 23938748
retrovirus; Mendelian inheritance; germ-line infection; receptors; tropism
7.  Influence of Duffy antigen receptor for chemokines on HIV infection 
Retrovirology  2013;10(Suppl 1):O31.
doi:10.1186/1742-4690-10-S1-O31
PMCID: PMC3848294
8.  Neutralizing antibodies to HIV-1 induced by immunization 
McCoy and Weiss discuss the generation of neutralizing antibodies to HIV induced by immunization.
Most neutralizing antibodies act at the earliest steps of viral infection and block interaction of the virus with cellular receptors to prevent entry into host cells. The inability to induce neutralizing antibodies to HIV has been a major obstacle to HIV vaccine research since the early days of the epidemic. However, in the past three years, the definition of a neutralizing antibody against HIV has been revolutionized by the isolation of extremely broad and potent neutralizing antibodies from HIV-infected individuals. Considerable hurdles remain for inducing neutralizing antibodies to a protective level after immunization. Meanwhile, novel technologies to bypass the induction of antibodies are being explored to provide prophylactic antibody-based interventions. This review addresses the challenge of inducing HIV neutralizing antibodies upon immunization and considers notable recent advances in the field. A greater understanding of the successes and failures for inducing a neutralizing response upon immunization is required to accelerate the development of an effective HIV vaccine.
doi:10.1084/jem.20121827
PMCID: PMC3570100  PMID: 23401570
9.  A Quantitative Meta-Analysis of Functional Imaging Studies of Social Rejection 
Scientific Reports  2013;3:2027.
Early neuroimaging studies using Cyberball suggested that social rejection activated the pain matrix, as identified in studies of physical pain. However, these early studies were characterized by small sample sizes. Our statistical multi-level kernel density analysis (MKDA) of Cyberball neuroimaging studies with 244 participants fails to support the claim that social rejection operates on the same pain matrix as nociceptive stimuli, questioning whether social pain is more figurative or literal. We also performed an MKDA of the neuroimaging studies of reliving a romantic rejection to test whether the pain matrix was activated if the rejection were more meaningful. Results again failed to support the notion that rejection activates the neural matrix identified in studies of physical pain. Reliving an unwanted rejection by a romantic partner was significantly characterized by activation within and beyond the “Cyberball” brain network, suggesting that the neural correlates of social pain are more complex than previously thought.
doi:10.1038/srep02027
PMCID: PMC3761131  PMID: 24002359
10.  Thirty years on: HIV receptor gymnastics and the prevention of infection 
BMC Biology  2013;11:57.
During 30 years of research on human immunodeficiency virus (HIV), our knowledge of its cellular receptors - CD4, CCR5 and CXCR4 - has illuminated aspects of the pathogenesis of the acquired immune deficiency syndrome (AIDS). Studying how the HIV envelope glycoproteins interact with the receptors led to anti-retroviral drugs based on blocking the docking or fusion of virus to the host cell. Genetic polymorphisms of CCR5 determine resistance to HIV infection and the rate of progression to AIDS. Eliciting neutralizing antibodies to the sites of receptor interaction on HIV glycoproteins is a promising approach to HIV vaccine development.
doi:10.1186/1741-7007-11-57
PMCID: PMC3660199  PMID: 23692808
AIDS; HIV; cell receptors; CD4; CCR5; CXCR4; Therapy
11.  A gp41 MPER-specific Llama VHH Requires a Hydrophobic CDR3 for Neutralization but not for Antigen Recognition 
PLoS Pathogens  2013;9(3):e1003202.
The membrane proximal external region (MPER) of the HIV-1 glycoprotein gp41 is targeted by the broadly neutralizing antibodies 2F5 and 4E10. To date, no immunization regimen in animals or humans has produced HIV-1 neutralizing MPER-specific antibodies. We immunized llamas with gp41-MPER proteoliposomes and selected a MPER-specific single chain antibody (VHH), 2H10, whose epitope overlaps with that of mAb 2F5. Bi-2H10, a bivalent form of 2H10, which displayed an approximately 20-fold increased affinity compared to the monovalent 2H10, neutralized various sensitive and resistant HIV-1 strains, as well as SHIV strains in TZM-bl cells. X-ray and NMR analyses combined with mutagenesis and modeling revealed that 2H10 recognizes its gp41 epitope in a helical conformation. Notably, tryptophan 100 at the tip of the long CDR3 is not required for gp41 interaction but essential for neutralization. Thus bi-2H10 is an anti-MPER antibody generated by immunization that requires hydrophobic CDR3 determinants in addition to epitope recognition for neutralization similar to the mode of neutralization employed by mAbs 2F5 and 4E10.
Author Summary
Due to the absence of an effective vaccine or cure for acquired immunodeficiency syndrome (AIDS), HIV-1 infections still result in high mortality. Two antibodies, 2F5 and 4E10, previously isolated from HIV-1 infected patients, prevent infections by binding to the MPER of gp41, a part of the virus that is difficult to access and only transiently exposed. Here, we immunized llamas with a gp41-based immunogen and subsequently isolated a small antibody fragment (VHH) that can easily access and recognize the MPER. We showed that a unit of two VHH, named bi-2H10, was indeed capable of preventing HIV-1 from infecting cells. We determined the three dimensional structure of the VHH and mapped its interaction site to an MPER region that overlaps with the 2F5 epitope. The 2H10 VHH displays a membrane binding component important for neutralization that resembles that of 2F5. In conclusion, we have developed an immunogen and a small antibody that may have great potential for development of novel anti-HIV/AIDS vaccines and treatments.
doi:10.1371/journal.ppat.1003202
PMCID: PMC3591319  PMID: 23505368
12.  Potent and broad neutralization of HIV-1 by a llama antibody elicited by immunization 
The Journal of Experimental Medicine  2012;209(6):1091-1103.
A heavy chain–only antibody isolated from a llama repeatedly immunized with trimeric HIV-1 Env neutralizes 96% of tested HIV-1 strains.
Llamas (Lama glama) naturally produce heavy chain–only antibodies (Abs) in addition to conventional Abs. The variable regions (VHH) in these heavy chain–only Abs demonstrate comparable affinity and specificity for antigens to conventional immunoglobulins despite their much smaller size. To date, immunizations in humans and animal models have yielded only Abs with limited ability to neutralize HIV-1. In this study, a VHH phagemid library generated from a llama that was multiply immunized with recombinant trimeric HIV-1 envelope proteins (Envs) was screened directly for HIV-1 neutralization. One VHH, L8CJ3 (J3), neutralized 96 of 100 tested HIV-1 strains, encompassing subtypes A, B, C, D, BC, AE, AG, AC, ACD, CD, and G. J3 also potently neutralized chimeric simian-HIV strains with HIV subtypes B and C Env. The sequence of J3 is highly divergent from previous anti–HIV-1 VHH and its own germline sequence. J3 achieves broad and potent neutralization of HIV-1 via interaction with the CD4-binding site of HIV-1 Env. This study may represent a new benchmark for immunogens to be included in B cell–based vaccines and supports the development of VHH as anti–HIV-1 microbicides.
doi:10.1084/jem.20112655
PMCID: PMC3371729  PMID: 22641382
13.  Potent Autologous and Heterologous Neutralizing Antibody Responses Occur in HIV-2 Infection across a Broad Range of Infection Outcomes 
Journal of Virology  2012;86(2):930-946.
Few studies have explored the role of neutralizing antibody (NAb) responses in controlling HIV-2 viremia and disease progression. Using a TZM-bl neutralization assay, we assessed heterologous and autologous NAb responses from a community cohort of HIV-2-infected individuals with a broad range of disease outcomes in rural Guinea-Bissau. All subjects (n = 40) displayed exceptionally high heterologous NAb titers (50% inhibitory plasma dilution or 50% inhibitory concentration [IC50], 1:7,000 to 1:1,000,000) against 5 novel primary HIV-2 envelopes and HIV-2 7312A, whereas ROD A and 3 primary envelopes were relatively resistant to neutralization. Most individuals also showed high autologous NAb against contemporaneous envelopes (78% of plasma-envelope combinations in 69 envelopes from 21 subjects), with IC50s above 1:10,000. No association between heterologous or autologous NAb titer and greater control of HIV-2 was found. A subset of envelopes was found to be more resistant to neutralization (by plasma and HIV-2 monoclonal antibodies). These envelopes were isolated from individuals with greater intrapatient sequence diversity and were associated with changes in potential N-linked glycosylation sites but not CD4 independence or CXCR4 use. Plasma collected from up to 15 years previously was able to potently neutralize recent autologous envelopes, suggesting a lack of escape from NAb and the persistence of neutralization-sensitive variants over time, despite significant NAb pressure. We conclude that despite the presence of broad and potent NAb responses in HIV-2-infected individuals, these are not the primary forces behind the dichotomous outcomes observed but reveal a limited capacity for adaptive selection and escape from host immunity in HIV-2 infection.
doi:10.1128/JVI.06126-11
PMCID: PMC3255814  PMID: 22072758
14.  100 years of Rous sarcoma virus 
The Journal of Experimental Medicine  2011;208(12):2351-2355.
In celebration of the centennial of the publication of Peyton Rous’s JEM paper on Rous sarcoma virus, this Perspective illustrates Rous’s broad and long-lasting influence on studies of tumor virology, oncogenes, and the molecular basis of carcinogenesis.
The discovery of Rous sarcoma virus, which was reported by Peyton Rous in the Journal of Experimental Medicine 100 years ago, opened the field of tumor virology. It showed that some cancers have infectious etiology, led to the discovery of oncogenes, and laid the foundation for the molecular mechanisms of carcinogenesis. Rous spent his entire research career at The Rockefeller Institute, and he was the JEM’s longest serving editor. Here, we comment briefly on the life of this remarkable scientist and on the importance of his discoveries.
doi:10.1084/jem.20112160
PMCID: PMC3256973  PMID: 22110182
15.  Duffy-Null–Associated Low Neutrophil Counts Influence HIV-1 Susceptibility in High-Risk South African Black Women 
The Duffy-null trait and ethnic neutropenia are highly prevalent in Africa. The authors found that the trait of Duffy-null–associated low neutrophil counts associated with increased HIV-1 susceptibility. The possible contribution of this trait to the high prevalence of HIV-1 in Africa requires further investigation
Background. The Duffy-null trait and ethnic netropenia are both highly prevalent in Africa. The influence of pre-seroconversion levels of peripheral blood cell counts (PBCs) on the risk of acquiring human immunodeficiency virus (HIV)–1 infection among Africans is unknown.
Methods. The triangular relationship among pre-seroconversion PBC counts, host genotypes, and risk of HIV acquisition was determined in a prospective cohort of black South African high-risk female sex workers. Twenty-seven women had seroconversion during follow-up, and 115 remained HIV negative for 2 years, despite engaging in high-risk activity.
Results. Pre-seroconversion neutrophil counts in women who subsequently had seroconversion were significantly lower, whereas platelet counts were higher, compared with those who remained HIV negative. Comprising 27% of the cohort, subjects with pre-seroconversion neutrophil counts of <2500 cells/mm3 had a ∼3-fold greater risk of acquiring HIV infection. In a genome-wide association analyses, an African-specific polymorphism (rs2814778) in the promoter of Duffy Antigen Receptor for Chemokines (DARC −46T > C) was significantly associated with neutrophil counts (P = 7.9 × 10−11). DARC −46C/C results in loss of DARC expression on erthyrocytes (Duffy-null) and resistance to Plasmodium vivax malaria, and in our cohort, only subjects with this genotype had pre-seroconversion neutrophil counts of <2500 cells/mm3. The risk of acquiring HIV infection was ∼3-fold greater in those with the trait of Duffy-null–associated low neutrophil counts, compared with all other study participants.
Conclusions. Pre-seroconversion neutrophil and platelet counts influence risk of HIV infection. The trait of Duffy-null–associated low neutrophil counts influences HIV susceptibility. Because of the high prevalence of this trait among persons of African ancestry, it may contribute to the dynamics of the HIV epidemic in Africa.
doi:10.1093/cid/cir119
PMCID: PMC3115278  PMID: 21507922
16.  Llama Antibody Fragments Recognizing Various Epitopes of the CD4bs Neutralize a Broad Range of HIV-1 Subtypes A, B and C 
PLoS ONE  2012;7(3):e33298.
Many of the neutralising antibodies, isolated to date, display limited activities against the globally most prevalent HIV-1 subtypes A and C. Therefore, those subtypes are considered to be an important target for antibody-based therapy. Variable domains of llama heavy chain antibodies (VHH) have some superior properties compared with classical antibodies. Therefore we describe the application of trimeric forms of envelope proteins (Env), derived from HIV-1 of subtype A and B/C, for a prolonged immunization of two llamas. A panel of VHH, which interfere with CD4 binding to HIV-1 Env were selected with use of panning. The results of binding and competition assays to various Env, including a variant with a stabilized CD4-binding state (gp120Ds2), cross-competition experiments, maturation analysis and neutralisation assays, enabled us to classify the selected VHH into three groups. The VHH of group I were efficient mainly against viruses of subtype A, C and B′/C. The VHH of group II resemble the broadly neutralising antibody (bnmAb) b12, neutralizing mainly subtype B and C viruses, however some had a broader neutralisation profile. A representative of the third group, 2E7, had an even higher neutralization breadth, neutralizing 21 out of the 26 tested strains belonging to the A, A/G, B, B/C and C subtypes. To evaluate the contribution of certain amino acids to the potency of the VHH a small set of the mutants were constructed. Surprisingly this yielded one mutant with slightly improved neutralisation potency against 92UG37.A9 (subtype A) and 96ZM651.02 (subtype C). These findings and the well-known stability of VHH indicate the potential application of these VHH as anti-HIV-1 microbicides.
doi:10.1371/journal.pone.0033298
PMCID: PMC3305327  PMID: 22438910
17.  Llama-Derived Single Domain Antibodies to Build Multivalent, Superpotent and Broadened Neutralizing Anti-Viral Molecules 
PLoS ONE  2011;6(4):e17665.
For efficient prevention of viral infections and cross protection, simultaneous targeting of multiple viral epitopes is a powerful strategy. Llama heavy chain antibody fragments (VHH) against the trimeric envelope proteins of Respiratory Syncytial Virus (Fusion protein), Rabies virus (Glycoprotein) and H5N1 Influenza (Hemagglutinin 5) were selected from llama derived immune libraries by phage display. Neutralizing VHH recognizing different epitopes in the receptor binding sites on the spikes with affinities in the low nanomolar range were identified for all the three viruses by viral neutralization assays. By fusion of VHH with variable linker lengths, multimeric constructs were made that improved neutralization potencies up to 4,000-fold for RSV, 1,500-fold for Rabies virus and 75-fold for Influenza H5N1. The potencies of the VHH constructs were similar or better than best performing monoclonal antibodies. The cross protection capacity against different viral strains was also improved for all three viruses, both by multivalent (two or three identical VHH) and biparatopic (two different VHH) constructs. By combining a VHH neutralizing RSV subtype A, but not subtype B with a poorly neutralizing VHH with high affinity for subtype B, a biparatopic construct was made with low nanomolar neutralizing potency against both subtypes. Trivalent anti-H5N1 VHH neutralized both Influenza H5N1 clade1 and 2 in a pseudotype assay and was very potent in neutralizing the NIBRG-14 Influenza H5N1 strain with IC50 of 9 picomolar. Bivalent and biparatopic constructs against Rabies virus cross neutralized both 10 different Genotype 1 strains and Genotype 5.
The results show that multimerization of VHH fragments targeting multiple epitopes on a viral trimeric spike protein is a powerful tool for anti-viral therapy to achieve “best-in-class” and broader neutralization capacity.
doi:10.1371/journal.pone.0017665
PMCID: PMC3069976  PMID: 21483777
18.  Extensive complement-dependent enhancement of HIV-1 by autologous non-neutralising antibodies at early stages of infection 
Retrovirology  2011;8:16.
Background
Non-neutralising antibodies to the envelope glycoprotein are elicited during acute HIV-1 infection and are abundant throughout the course of disease progression. Although these antibodies appear to have negligible effects on HIV-1 infection when assayed in standard neutralisation assays, they have the potential to exert either inhibitory or enhancing effects through interactions with complement and/or Fc receptors. Here we report that non-neutralising antibodies produced early in response to HIV-1 infection can enhance viral infectivity.
Results
We investigated this complement-mediated antibody-dependent enhancement (C'-ADE) of early HIV infection by carrying out longitudinal studies with primary viruses and autologous sera derived sequentially from recently infected individuals, using a T cell line naturally expressing the complement receptor 2 (CR2; CD21). The C'-ADE was consistently observed and in some cases achieved infection-enhancing levels of greater than 350-fold, converting a low-level infection to a highly destructive one. C'-ADE activity declined as a neutralising response to the early virus emerged, but later virus isolates that had escaped the neutralising response demonstrated an increased capacity for enhanced infection by autologous antibodies. Moreover, sera with autologous enhancing activity were capable of C'ADE of heterologous viral isolates, suggesting the targeting of conserved epitopes on the envelope glycoprotein. Ectopic expression of CR2 on cell lines expressing HIV-1 receptors was sufficient to render them sensitive to C'ADE.
Conclusions
Taken together, these results suggest that non-neutralising antibodies to the HIV-1 envelope that arise during acute infection are not 'passive', but in concert with complement and complement receptors may have consequences for HIV-1 dissemination and pathogenesis.
doi:10.1186/1742-4690-8-16
PMCID: PMC3065417  PMID: 21401915
20.  Crystal Structure and Size-Dependent Neutralization Properties of HK20, a Human Monoclonal Antibody Binding to the Highly Conserved Heptad Repeat 1 of gp41 
PLoS Pathogens  2010;6(11):e1001195.
The human monoclonal antibody (mAb) HK20 neutralizes a broad spectrum of primary HIV-1 isolates by targeting the highly conserved heptad repeat 1 (HR1) of gp41, which is transiently exposed during HIV-1 entry. Here we present the crystal structure of the HK20 Fab in complex with a gp41 mimetic 5-Helix at 2.3 Å resolution. HK20 employs its heavy chain CDR H2 and H3 loops to bind into a conserved hydrophobic HR1 pocket that is occupied by HR2 residues in the gp41 post fusion conformation. Compared to the previously described HR1-specific mAb D5, HK20 approaches its epitope with a different angle which might favor epitope access and thus contribute to its higher neutralization breadth and potency. Comparison of the neutralization activities of HK20 IgG, Fab and scFv employing both single cycle and multiple cycle neutralization assays revealed much higher potencies for the smaller Fab and scFv over IgG, implying that the target site is difficult to access for complete antibodies. Nevertheless, two thirds of sera from HIV-1 infected individuals contain significant titers of HK20-inhibiting antibodies. The breadth of neutralization of primary isolates across all clades, the higher potencies for C-clade viruses and the targeting of a distinct site as compared to the fusion inhibitor T-20 demonstrate the potential of HK20 scFv as a therapeutic tool.
Author Summary
The HIV-1 envelope glycoprotein composed of the receptor binding subunit gp120 and the fusion protein gp41 is the prime target for neutralizing antibodies. Receptor binding induces a conformational change in gp41 that transiently exposes the conserved heptad repeat 1 (HR1) region. We have previously isolated the human HR1-specific mAb HK20 and provide now the structural basis for epitope recognition. HK20 employs mainly its CDR H2 and H3 for binding similar to HR1 binding of mAb D5. We demonstrate that HK20 and D5 bind HR1 with similar affinities; however, HK20 has a broader neutralization breadth than D5, which might be due to the differences in their approach angles of epitope recognition. Competition analyses of 33 sera from HIV-1 infected individuals reveal significant titers of HK20-inhibiting antibodies in 20 cases, confirming the immunogenicity of the epitope. We demonstrate further that HK20 IgG have limited neutralization breadth and potency while smaller HK20 Fabs and scFv reveal a broad cross clade neutralization breadth. This suggests that the accessibility of the HR1 epitope limits the value of HR1 mAbs for infection prevention, but highlights the importance of smaller versions such Fabs or scFv to combat infection alone or in synergistic approaches with other antivirals.
doi:10.1371/journal.ppat.1001195
PMCID: PMC2987821  PMID: 21124990
21.  A cautionary tale of virus and disease 
BMC Biology  2010;8:124.
The recent identification of the gammaretrovirus XMRV and a second gammaretrovirus of a different subtype in chronic fatigue syndrome has aroused much interest, not least among sufferers. However, it remains highly controversial whether the detection of these viruses represents true infection or laboratory artifacts.
doi:10.1186/1741-7007-8-124
PMCID: PMC2946284  PMID: 20920148
22.  Crystal Structure of the Neutralizing Llama VHH D7 and Its Mode of HIV-1 gp120 Interaction 
PLoS ONE  2010;5(5):e10482.
HIV-1 entry into host cells is mediated by the sequential binding of the envelope glycoprotein gp120 to CD4 and a chemokine receptor. Antibodies binding to epitopes overlapping the CD4-binding site on gp120 are potent inhibitors of HIV entry, such as the llama heavy chain antibody fragment VHH D7, which has cross-clade neutralizing properties and competes with CD4 and mAb b12 for high affinity binding to gp120. We report the crystal structure of the D7 VHH at 1.5 Å resolution, which reveals the molecular details of the complementarity determining regions (CDR) and substantial flexibility of CDR3 that could facilitate an induced fit interaction with gp120. Structural comparison of CDRs from other CD4 binding site antibodies suggests diverse modes of interaction. Mutational analysis identified CDR3 as a key component of gp120 interaction as determined by surface plasmon resonance. A decrease in affinity is directly coupled to the neutralization efficiency since mutations that decrease gp120 interaction increase the IC50 required for HIV-1 IIIB neutralization. Thus the structural study identifies the long CDR3 of D7 as the key determinant of interaction and HIV-1 neutralization. Furthermore, our data confirm that the structural plasticity of gp120 can accommodate multiple modes of antibody binding within the CD4 binding site.
doi:10.1371/journal.pone.0010482
PMCID: PMC2864739  PMID: 20463957
23.  Generation of a Family-specific Phage Library of Llama Single Chain Antibody Fragments That Neutralize HIV-1* 
The Journal of Biological Chemistry  2010;285(25):19116-19124.
Recently, we described llama antibody fragments (VHH) that can neutralize human immunodeficiency virus, type 1 (HIV-1). These VHH were obtained after selective elution of phages carrying an immune library raised against gp120 of HIV-1 subtype B/C CN54 with soluble CD4. We describe here a new, family-specific approach to obtain the largest possible diversity of related VHH that compete with soluble CD4 for binding to the HIV-1 envelope glycoprotein. The creation of this family-specific library of homologous VHH has enabled us to isolate phages carrying similar nucleotide sequences as the parental VHH. These VHH displayed varying binding affinities and neutralization phenotypes to a panel of different strains and subtypes of HIV-1. Sequence analysis of the homologs showed that the C-terminal three amino acids of the CDR3 loop were crucial in determining the specificity of these VHH for different subtype C HIV-1 strains. There was a positive correlation between affinity of VHH binding to gp120 of HIV-1 IIIB and the breadth of neutralization of diverse HIV-1 envelopes. The family-specific approach has therefore allowed us to better understand the interaction of the CD4-binding site antibodies with virus strain specificity and has potential use for the bioengineering of antibodies and HIV-1 vaccine development.
doi:10.1074/jbc.M110.116699
PMCID: PMC2885190  PMID: 20400507
Antibodies; HIV; Human Immunodeficiency Virus; Kinetics; Retrovirus; Surface Plasmon Resonance; VHH; Llama Heavy Chain Antibodies; Neutralizing Antibodies; Phage Display
24.  Heterosubtypic neutralizing antibodies are produced by individuals immunized with a seasonal influenza vaccine 
The Journal of Clinical Investigation  2010;120(5):1663-1673.
The target of neutralizing antibodies that protect against influenza virus infection is the viral protein HA. Genetic and antigenic variation in HA has been used to classify influenza viruses into subtypes (H1–H16). The neutralizing antibody response to influenza virus is thought to be specific for a few antigenically related isolates within a given subtype. However, while heterosubtypic antibodies capable of neutralizing multiple influenza virus subtypes have been recently isolated from phage display libraries, it is not known whether such antibodies are produced in the course of an immune response to influenza virus infection or vaccine. Here we report that, following vaccination with seasonal influenza vaccine containing H1 and H3 influenza virus subtypes, some individuals produce antibodies that cross-react with H5 HA. By immortalizing IgG-expressing B cells from 4 individuals, we isolated 20 heterosubtypic mAbs that bound and neutralized viruses belonging to several HA subtypes (H1, H2, H5, H6, and H9), including the pandemic A/California/07/09 H1N1 isolate. The mAbs used different VH genes and carried a high frequency of somatic mutations. With the exception of a mAb that bound to the HA globular head, all heterosubtypic mAbs bound to acid-sensitive epitopes in the HA stem region. Four mAbs were evaluated in vivo and protected mice from challenge with influenza viruses representative of different subtypes. These findings reveal that seasonal influenza vaccination can induce polyclonal heterosubtypic neutralizing antibodies that cross-react with the swine-origin pandemic H1N1 influenza virus and with the highly pathogenic H5N1 virus.
doi:10.1172/JCI41902
PMCID: PMC2860935  PMID: 20389023
25.  Analysis of Memory B Cell Responses and Isolation of Novel Monoclonal Antibodies with Neutralizing Breadth from HIV-1-Infected Individuals 
PLoS ONE  2010;5(1):e8805.
Background
The isolation of human monoclonal antibodies (mAbs) that neutralize a broad spectrum of primary HIV-1 isolates and the characterization of the human neutralizing antibody B cell response to HIV-1 infection are important goals that are central to the design of an effective antibody-based vaccine.
Methods and Findings
We immortalized IgG+ memory B cells from individuals infected with diverse clades of HIV-1 and selected on the basis of plasma neutralization profiles that were cross-clade and relatively potent. Culture supernatants were screened using various recombinant forms of the envelope glycoproteins (Env) in multiple parallel assays. We isolated 58 mAbs that were mapped to different Env surfaces, most of which showed neutralizing activity. One mAb in particular (HJ16) specific for a novel epitope proximal to the CD4 binding site on gp120 selectively neutralized a multi-clade panel of Tier-2 HIV-1 pseudoviruses, and demonstrated reactivity that was comparable in breadth, but distinct in neutralization specificity, to that of the other CD4 binding site-specific neutralizing mAb b12. A second mAb (HGN194) bound a conserved epitope in the V3 crown and neutralized all Tier-1 and a proportion of Tier-2 pseudoviruses tested, irrespective of clade. A third mAb (HK20) with broad neutralizing activity, particularly as a Fab fragment, recognized a highly conserved epitope in the HR-1 region of gp41, but showed striking assay-dependent selectivity in its activity.
Conclusions
This study reveals that by using appropriate screening methods, a large proportion of memory B cells can be isolated that produce mAbs with HIV-1 neutralizing activity. Three of these mAbs show unusual breadth of neutralization and therefore add to the current panel of HIV-1 neutralizing antibodies with potential for passive protection and template-based vaccine design.
doi:10.1371/journal.pone.0008805
PMCID: PMC2808385  PMID: 20098712

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