Epitope Substitutions Research Articles

Resistance Analysis Following Sotrovimab Treatment in Participants with COVID-19 During the Phase III COMET-ICE Study

Aim: Sotrovimab is an engineered human monoclonal antibody that binds a conserved region of the SARS-CoV-2 spike protein. The COMET-ICE phase III study evaluated sotrovimab for treatment of mild to moderate COVID-19 in nonhospitalized participants with ≥1 risk factor for severe disease progression. Materials & methods: We evaluated the presence of circulating SARS-CoV-2 variants of concern or interest (VOCs/VOIs) and characterized the presence of baseline, post-baseline and emergent amino acid substitutions detected in the epitope of sotrovimab in SARS-CoV-2. Results: None of the sotrovimab-treated participants with baseline epitope substitutions, and 1 of 48 sotrovimab-treated participants with post-baseline epitope substitutions, met the primary clinical endpoint for progression. Conclusion: Overall, progression was not associated with identified VOC/VOI or the presence of epitope substitutions in sotrovimab-treated participants.

304. Viral resistance analysis in the COMET-PEAK study: sotrovimab treatment in participants with mild-to-moderate COVID-19

Abstract Background Sotrovimab is a human monoclonal antibody targeting a conserved region of the SARS-CoV-2 spike (S) protein. COMET-PEAK was a 3-part, phase 2 study that evaluated intravenous (500 mg) and intramuscular (500 mg and 250 mg) administration of sotrovimab in outpatients (n=353) with mild-to-moderate COVID-19. We assessed amino acid substitutions in the SARS-CoV-2 S protein and circulating variants of concern/interest (VOC/VOI) in COMET-PEAK participants (enrolled Feb–July 2021). Methods Mid-turbinate (Part A) or nasopharyngeal (Part B/C) samples were obtained from all participants at Baseline and Post-Baseline visits. Next generation sequencing of the SARS-CoV-2 S gene was conducted using Illumina MiSeq with a ≥5% allelic frequency cut-off for samples with a viral load above 3.0 log10 copies/mL. Baseline, post-baseline and treatment-emergent (TE) substitutions were assessed, and prevalence of VOC/VOI was evaluated. Phenotypic analyses of epitope substitutions were conducted using a pseudotyped virus assay. Results In total, 282/353 participants had sequencing results for ≥1 visit (253 baseline, 248 post-baseline), and 219 had paired baseline and post-baseline sequences; among these, 149 (68%) had TE substitutions in the S protein (26 [12%] in the epitope). E340K was the predominant TE substitution in the epitope (15 [7%]). Across all arms 92/245 (38%) experienced virologic rebound, 8 of whom (Part A: 2; Part B: 2; Part C: 4) had TE substitutions in the epitope; none had evidence of clinical progression to severe disease. Prevalence of VOC/VOI or single amino acid substitutions of concern was 94% (266/282); the most frequent were Alpha (Part A: 8/16 [50%]; Part B: 75/128 [59%]) and Delta (Part C: 99/122 [81%]). Of 7 participants with evidence of clinical progression, none had S protein substitutions in the epitope and all had VOC/VOI (3 Alpha, 3 Delta, 1 Gamma). Sotrovimab effectively neutralized most epitope substitutions tested in vitro; P337L and E340A/K/V conferred significantly reduced susceptibility. Conclusion There was no evidence that sotrovimab epitope substitutions were associated with clinical progression or virologic rebound. These data are consistent with those from the COMET-ICE study. Funding Vir Biotechnology/GSK (NCT04779879). Disclosures Phillip J Yates, PhD, GlaxoSmithKline: Employee Jennifer Moore, MD, GlaxoSmithKline: Employee Jennifer Han, MD, GlaxoSmithKline: Employee Andrew Skingsley, MD, GlaxoSmithKline: Employee|GlaxoSmithKline: Stocks/Bonds Gretja Schnell, PhD, Vir Biotechnology: Employee|Vir Biotechnology: Stocks/Bonds Andrea L. Cathcart, PhD, Vir Biotechnology: Employee|Vir Biotechnology: Stocks/Bonds Melissa Aldinger, PharmD, Vir Biotechnology: Employee|Vir Biotechnology: Stocks/Bonds Amanda Peppercorn, MD, GlaxoSmithKline: Employee|GlaxoSmithKline: Stocks/Bonds Jill Walker, PhD, GlaxoSmithKline: Employee.

1150. Resistance Analysis in the COMET-TAIL Study: Participants with Mild-to-Moderate COVID-19 Treated with Intramuscular or Intravenous Sotrovimab

Abstract Background Sotrovimab (VIR-7831) is an engineered human monoclonal antibody targeting a conserved region of the SARS-CoV-2 spike protein; it has been shown to have a favorable safety profile and be effective for early treatment of high-risk COVID-19 patients. The COMET-TAIL phase 3 study evaluated sotrovimab administered intravenously (IV) or intramuscularly (IM) for the treatment of participants with mild to moderate COVID-19 who are at high risk of disease progression. Methods Between June to August 2021, 973 participants were randomized and received sotrovimab by 500 mg IV infusion or by 500 or 250 mg IM injection. Deep sequencing of the spike gene was performed on nasopharyngeal samples. Baseline (BL; Day 1 or Day 3), post-BL (Day 5 or later), treatment-emergent (TE) substitutions at sotrovimab epitope positions, and presence of variants of concern/interest (VOC/VOI), were evaluated at a ≥5% allelic frequency. Phenotypic analyses were conducted using a pseudotyped virus assay. Results Sequences were available from 764 participants (500 mg IV: 314/393; 500 mg IM: 302/387; 250 mg IM: 148/193). Consistent with VOC circulation during enrollment, the Delta variant was detected in 88.2% (674/764) of participants. Alpha and Mu variants were also seen at >2% prevalence. Of the 764 participants, 26 met the primary endpoint for clinical progression to hospitalization >24 hours or death due to any cause through day 29 and were infected with Delta (500 mg IV: 4; 500 mg IM: 9; 250 mg IM: 11), Alpha (500 mg IM: 1), or Mu (500 mg IV: 1) variants. Substitutions at sotrovimab epitope positions were similar across arms and were detected in 82/764 (10.7%) participants at any visit (500 mg IV: 42/314; 500 mg IM: 27/302; 250 mg IM: 13/148). Of these, 2 participants experienced clinical progression: 1 participant infected with the Mu variant (500 mg IV) carried the characteristic R346K substitution at BL; 1 participant infected with the Delta variant (500 mg IM) had P337L and E340K substitutions detected at Day 3 and P337L was enriched at Day 8. The predominant TE epitope substitutions included P337L and E340A/K/V, which confer reduced susceptibility to sotrovimab in vitro. Conclusion Overall, TE epitope substitutions were not associated with clinical progression. Funding Vir & GSK (NCT04913675) Disclosures Maria L. Agostini, PhD, Vir Biotechnology, Inc: Employee|Vir Biotechnology, Inc: Stocks/Bonds Gretja Schnell, PhD, Vir Biotechnology: Employee|Vir Biotechnology: Stocks/Bonds Julia di Iulio, PharmD, PhD, MBA, Vir Biotechnology: Employee|Vir Biotechnology: Stocks/Bonds Anita Kohli, MD, GlaxoSmithKline: Third party funding to Vir|Vir Biotechnology Inc: Support as a trial site paid to my institution and non-financial support for medical writing support Adrienne E. Shapiro, MD, PhD, Vir Biotechnology: Support as a trial site paid to my institution and non-financial support for medical writing GlaxoSmithKline third party funding to Vir support Elias H. Sarkis, MD, Abbvie: Grant/Research Support|Abbvie: Speakers Bureau|Eisai: Grant/Research Support|GlaxoSmithKline: Third party funding to Vir|Ironshore: Grant/Research Support|Janssen: Speakers Bureau|Lilly: Grant/Research Support|Otsuka: Grant/Research Support|Teva: Speakers Bureau|Vir Biotechnology Inc: Support as a trial site paid to my institution and non-financial support for medical writing support Dave Inman, MSc, GlaxoSmithKline: Employee|GlaxoSmithKline: Stocks/Bonds Amanda Peppercorn, MD, GlaxoSmithKline: Employee|GlaxoSmithKline: Stocks/Bonds Andrew Skingsley, MD, GlaxoSmithKline: Employee|GlaxoSmithKline: Stocks/Bonds Leah A. Gaffney, PhD, Vir Biotechnology: Employee|Vir Biotechnology: Stocks/Bonds Melissa Aldinger, PharmD, Vir Biotechnology: Employee|Vir Biotechnology: Stocks/Bonds Christy M. Hebner, PhD, Vir Biotechnology: Employee|Vir Biotechnology: Stocks/Bonds Andrea L. Cathcart, PhD, Vir Biotechnology: Employee|Vir Biotechnology: Stocks/Bonds.

Multiepitope Fusion Antigen: MEFA, an Epitope- and Structure-Based Vaccinology Platform for Multivalent Vaccine Development.

Vaccines are regarded as the most cost-effective countermeasure against infectious diseases. One challenge often affecting vaccine development is antigenic diversity or pathogen heterogeneity. Different strains produce immunologically heterogeneous virulence factors, therefore an effective vaccine needs to induce broad-spectrum host immunity to provide cross-protection. Recent advances in genomics and proteomics, particularly computational biology and structural biology, establishes structural vaccinology and highlights the feasibility of developing effective and precision vaccines. Here, we introduce the epitope- and structure-based vaccinology platform multiepitope-fusion-antigen (MEFA), and provide instructions to generate polyvalent MEFA immunogens for vaccine development. Conceptually, MEFA combines epitope vaccinology and structural vaccinology to enable a protein immunogen to present heterogeneous antigenic domains (epitopes) and to induce broadly protective immunity against different virulence factors, strains or diseases. Methodologically, the MEFA platform first identifies a safe, structurally stable and strongly immunogenic backbone protein and immunodominant (ideally neutralizing or protective) epitopes from heterogeneous strains or virulence factors of interest. Then, assisted with protein modeling and molecule dynamic simulation, MEFA integrates heterogeneous epitopes into a backbone protein via epitope substitution for a polyvalentMEFA protein and mimics epitope native antigenicity. Finally, the MEFA protein is examined for broad immunogenicity in animal immunization, and assessed for potential application for multivalent vaccine development in preclinical studies.

Mapping antibody binding using multiplexed epitope substitution analysis

A more complete understanding of antibody epitopes would aid the development of diagnostics, therapeutic antibodies, and vaccines. However, current methods for mapping antibody binding to epitopes require a targeted experimental approach, which limits throughput. To address these limitations, we developed Multiplexed Epitope Substitution Analysis (MESA) which can rapidly characterize various distinct epitopes using millions of antibody-binding peptides. We screened peptides from a random 12-mer library that bound to human serum antibody repertoires and determined their sequences using next-generation sequencing (NGS). Computationally, we divided target epitope sequences into overlapping k-mer subsequences and substituted the positions in each k-mer with all 20 amino acids, mimicking a saturation mutagenesis. We then determined enrichments of the substituted k-mers in the screened peptide dataset and used these enrichments to identify substitutions favored for binding at each position in the target epitope, ultimately revealing the precise binding motif. To validate MESA, we determined binding motifs for monoclonal antibodies spiked into serum, recovering the expected binding positions and amino acid preferences. To characterize epitopes bound by a population, we analyzed 50 serum specimens to determine the binding motifs within various target epitopes from common pathogens. Additionally, by analyzing various HSV-1 glycoprotein epitopes, MESA revealed unique binding signatures for HSV-1 seropositive specimens and demonstrated the variability of binding signatures within a population. These results demonstrate that MESA can rapidly identify and characterize binding motifs for an unlimited number of epitopes from a single experiment, accelerating discoveries and enhancing our understanding of antibody-epitope interactions.

Functional role of surface layer proteins of Lactobacillus acidophilus L-92 in stress tolerance and binding to host cell proteins.

Lactobacillus acidophilus surface layer proteins (SLPs) self-assemble into a monolayer that is non-covalently bound to the outer surface of the cells. There they are in direct contact with the environment, environmental stressors and gut components of the host in which the organism resides. The role of L. acidophilus SLPs is not entirely understood, although SLPs seem to be essential for bacterial growth. We constructed three L. acidophilus L-92 strains, each expressing a mutant of the most abundant SLP, SlpA. Each carried a 12-amino acid c-myc epitope substitution at a different position in the protein. A strain was also obtained that expressed the SlpA paralog SlpB from an originally silent slpB gene. All four strains behaved differently with respect to growth under various stress conditions, such as the presence of salt, ox gall or ethanol, suggesting that SlpA affects stress tolerance in L. acidophilus L-92. Also, the four mutants showed differential in vitro binding ability to human host cell proteins such as uromodulin or dendritic cell (DC)-specific intercellular adhesion molecule-3 grabbing non-integrin (DC-SIGN). Furthermore, co-culture of murine immature DCs with a mutant strain expressing one of the recombinant SlpA proteins changed the concentrations of the cytokines IL-10 and IL-12. Our data suggest that SlpA and SlpB of L. acidophilus participate in bacterial stress tolerance and binding to uromodulin or DC-SIGN, possibly leading to effective immune-modification.

Spread and evolution of influenza A (H1N1) pdm09 virus in Hubei, 2015–2019

ObjectiveTo understand spread, phylogenetic characteristics, epitope and drug-resistance site of influenza A (H1N1) pdm09 virus in Hubei province during 2015–2019. MethodsThe spread period of the virus were divided according to the positive rate of influenza virus nucleic acid detection downloaded from influenza surveillance information system in China. Sequencing of 39 strains of influenza A (H1N1) pdm09 virus isolated from the positive samples in real-time fluorescence PCR in Hubei during 2015–2019 was conducted and 13 sequences of the strains isolated from Hubei were downloaded from global influenza database to analyze the distribution of phylogenetic clade, epitope and drug-resistant mutation sites by using bioinformatics software and simulate the structure of mutation sites by 3D modeling. ResultsThe annual spread of influenza A (H1N1) pdm09 virus lasted for 11–14 weeks in Hubei from 2015 to 2019, and the spread intensity increased year by year. Multiple branches of 6B.1A to 6B.1A7 evolved from 6B clade in phylogenetic tree of the HA and NA genes. Twelve epitope substitutions of HA and two drug-resistance sites of NA were detected, and there were obvious differences in D222G substitution virulence identifying marker and drug-resistance site I223V on homology in 3D modeling structure. ConclusionThe spread intensity and phylogenetic clade number of influenza A (H1N1) pdm09 virus increased In Hubei during 2015–2019. Sporadic virulence marker and drug-resistance site were detected for influenza A (H1N1) pdm09 virus. The results of this analysis can be used to improve the epidemiological research and genetic evolution surveillance of influenza virus in Hubei.

Molecular Basis for Necitumumab Inhibition of EGFR Variants Associated with Acquired Cetuximab Resistance.

Acquired resistance to cetuximab, an antibody that targets the EGFR, impacts clinical benefit in head and neck, and colorectal cancers. One of the mechanisms of resistance to cetuximab is the acquisition of mutations that map to the cetuximab epitope on EGFR and prevent drug binding. We find that necitumumab, another FDA-approved EGFR antibody, can bind to EGFR that harbors the most common cetuximab-resistant substitution, S468R (or S492R, depending on the amino acid numbering system). We determined an X-ray crystal structure to 2.8 Å resolution of the necitumumab Fab bound to an S468R variant of EGFR domain III. The arginine is accommodated in a large, preexisting cavity in the necitumumab paratope. We predict that this paratope shape will be permissive to other epitope substitutions, and show that necitumumab binds to most cetuximab- and panitumumab-resistant EGFR variants. We find that a simple computational approach can predict with high success which EGFR epitope substitutions abrogate antibody binding. This computational method will be valuable to determine whether necitumumab will bind to EGFR as new epitope resistance variants are identified. This method could also be useful for rapid evaluation of the effect on binding of alterations in other antibody/antigen interfaces. Together, these data suggest that necitumumab may be active in patients who are resistant to cetuximab or panitumumab through EGFR epitope mutation. Furthermore, our analysis leads us to speculate that antibodies with large paratope cavities may be less susceptible to resistance due to mutations mapping to the antigen epitope. Mol Cancer Ther; 17(2); 521-31. ©2017 AACR.

Comparison of the Protective Efficacy of Neutralizing Epitopes of 2009 Pandemic H1N1 Influenza Hemagglutinin.

The 2009 H1N1 influenza (Pdm09) pandemic has been referred to as the first influenza pandemic of the twenty-first century. There is a marked difference in antigenicity between the pandemic H1N1 virus and past seasonal H1N1 viruses, which allowed the pandemic virus to spread rapidly in humans. Antibodies (Abs) against hemagglutinin (HA), especially neutralizing Abs against epitopes in the head of HA, play critical roles in defending the host against the virus. Some preexisting neutralizing Abs that recognize neutralizing epitopes of Pdm09 HA, thereby affording cross-protection, have been reported. To better understand the protective effects of epitopes in Pdm09 HA, we constructed a series of plasmid DNAs (DNA vaccines) by cloning various combinations of Pdm09 neutralizing epitopes into the HA backbone derived from A/PR/8/1934 (H1N1). We subsequently compared the protective immune responses induced by these various forms of HA in a mouse model. We found that the plasmid DNAs with epitope substitutions provided better protection against lethal virus challenge and induced higher strain-specific antibody titers, with epitope Sa being the most effective. Moreover, the combination of epitopes Sa and Sb provided almost complete protection in mice. These findings provide new insights into the protective efficacy of neutralizing epitopes of influenza HA.

Antibody fingerprints in lyme disease deciphered with high density peptide arrays.

Lyme disease is the most common tick-borne infectious disease in Europe and North America. Previous studies discovered the immunogenic role of a surface-exposed lipoprotein (VlsE) of Borreliella burgdorferi. We employed high density peptide arrays to investigate the antibody response to the VlsE protein in VlsE-positive patients by mapping the protein as overlapping peptides and subsequent in-depth epitope substitution analyses. These investigations led to the identification of antibody fingerprints represented by a number of key residues that are indispensable for the binding of the respective antibody. This approach allows us to compare the antibody specificities of different patients to the resolution of single amino acids. Our study revealed that the sera of VlsE-positive patients recognize different epitopes on the protein. Remarkably, in those cases where the same epitope is targeted, the antibody fingerprint is almost identical. Furthermore, we could correlate two fingerprints with human autoantigens and an Epstein-Barr virus epitope; yet, the link to autoimmune disorders seems unlikely and must be investigated in further studies. The other three fingerprints are much more specific for B. burgdorferi. Since antibody fingerprints of longer sequences have proven to be highly disease specific, our findings suggest that the fingerprints could function as diagnostic markers that can reduce false positive test results.

High‐frequency vaccine‐induced CD8+ T cells specific for an epitope naturally processed during infection with Mycobacterium tuberculosis do not confer protection

Relatively few MHC class I epitopes have been identified from Mycobacterium tuberculosis, but during the late stage of infection, CD8+ T‐cell responses to these epitopes are often primed at an extraordinary high frequency. Although clearly available for recognition during infection, their role in resistance to mycobacterial infections still remain unclear. As an alternative to DNA and viral vaccination platforms, we have exploited a novel CD8+ T‐cell‐inducing adjuvant, cationic adjuvant formulation 05 (dimethyldioctadecylammonium/trehalose dibehenate/poly (inositic:cytidylic) acid), to prime high‐frequency CD8 responses to the immunodominant H2‐Kb‐restricted IMYNYPAM epitope contained in the vaccine Ag tuberculosis (TB)10.4/Rv0288/ESX‐H (where ESX is mycobacterial type VII secretion system). We report that the amino acid C‐terminal to this minimal epitope plays a decisive role in proteasomal cleavage and epitope priming. The primary structure of TB10.4 is suboptimal for proteasomal processing of the epitope and amino acid substitutions in the flanking region markedly increased epitope‐specific CD8+ T‐cell responses. One of the optimized sequences was contained in the closely related TB10.3/Rv3019c/ESX‐R Ag and when recombinantly expressed and administered in the cationic adjuvant formulation 05 adjuvant, this Ag promoted very high CD8+ T‐cell responses. This abundant T‐cell response was functionally active but provided no protection against challenge, suggesting that CD8+ T cells play a limited role in protection against M. tuberculosis in the mouse model.

HBsAg Mutants Clustered Mainly Outside of “a” Determinant in Chronic Carriers From Azarbayjan Province, Iran

: Background: Hepatitis B virus (HBV) genetic and protein variations have been observed in chronic patients frequently. However, their role in the pathogenesis of chronicity has not been explored yet. Objectives: The aims of this study were to determine the genotypes as well as the patterns of variations distribution in chronically infected patients from the north western part of Iran. Materials and Methods: The surface genes from 17 chronic carriers were amplified, sequenced and subsequently aligned using international and national Iranian database. Results: All strains belonged to genotype D, subgenotype D1 and subtype ayw2. Of all 56 “mutations” that occurred at 37 nucleotide positions, 25 (44.6%) were missense (amino acid altering) and 31 (55.4%) were silent (no amino acid changing) (S/M ratio: 1.2). At the amino acid level, 21 (91.3%) out of 23 amino acid mutations occurred in different immune epitopes within the surface proteins, of which 3 (14.3%) occurred in B cell, 8 (38%) in T helper and 10 (47.7%) inside CTL epitopes. In general, the association between amino acid mutations and especially, immune epitope substitutions was more significant in terms of HBeAg status than ALT levels in patients. Conclusions: The distribution of amino acid mutations as well as the ratio between missense and silent nucleotide mutations (dN/dS) showed that a narrowly focused immune pressure had already been on the surface protein (especially CTL epitopes) which led to the emergence of escape mutants in these patients who were in tolerance phase of chronicity.

ScFv Antibody-induced Translocation of Cell-surface Heparan Sulfate Proteoglycan to Endocytic Vesicles

Cellular uptake of several viruses and polybasic macromolecules requires the expression of cell-surface heparan sulfate proteoglycan (HSPG) through as yet ill defined mechanisms. We unexpectedly found that among several cell-surface-binding single chain variable fragment (scFv) anti-HS antibody (alphaHS) clones, only one, AO4B08, efficiently translocated macromolecular cargo to intracellular vesicles through induction of HSPG endocytosis. Interestingly, AO4B08-induced PG internalization was strictly dependent on HS 2-O-sulfation and appeared independent of intact N-sulfation. AO4B08 and human immunodeficiency virus (HIV)-Tat, i.e. a well known cell-penetrating peptide, were shown to compete for the internalizing PG population. To obtain a more detailed characterization of this pathway, we have developed a procedure for the isolation of endocytic vesicles by conjugating AO4B08 with superparamagnetic nanoparticles. [(35)S]sulfate-labeled HSPG was found to accumulate in isolated, AO4B08-containing vesicles, providing the first biochemical evidence for intact HSPG co-internalization with its ligand. Further analysis revealed the existence of both syndecan, i.e. a transmembrane HSPG, and glycosyl-phosphatidyl-inositol-anchored glypican in purified vesicles. Importantly, internalized syndecan and glypican were found to co-localize in AO4B08-containing vesicles. Our data establish HSPGs as true internalizing receptors of macromolecular cargo and indicate that the sorting of cell-surface HSPG to endocytic vesicles is determined by a specific HS epitope that can be carried by both syndecan and glypican core protein.

Infectivity and Neutralization of Simian Immunodeficiency Virus with FLAG Epitope Insertion in gp120 Variable Loops

A FLAG epitope tag was substituted within variable loop 1 (V1), 2 (V2), or 4 (V4) of the gp120 envelope glycoprotein of simian immunodeficiency virus strain 239 (SIV239) to evaluate the extent to which each variable loop may serve as a target for antibody-mediated neutralization. Two sites within each variable loop of SIV239 were chosen for individual epitope tag insertions. FLAG epitope substitutions were also made in the V1, V2, and V4 loops of a neutralization-sensitive derivative of SIV239, SIV316. Of the 10 FLAG-tagged recombinant viruses analyzed, three (SIV239FV1b, SIV239FV2b, and SIV239FV4a) replicated with kinetics similar to those of the parental strain, SIV239, in both CEMx174 cells and the immortalized rhesus monkey T-cell line 221. The SIV316FV1b and SIV316FV4a FLAG variants replicated with a substantial lag, and the five remaining recombinants did not replicate detectably. Both gp160 and gp120 from replication-competent FLAG variants could be immunoprecipitated from transfected 293T cells by the anti-gp120 rhesus monoclonal antibody (RhMAb) 3.11H, the anti-FLAG MAb M2, and CD4-immunoglobulin, whereas only unprocessed gp160 was detected in 293T cells transfected with replication-defective variants. Furthermore, gp120 was detectably incorporated only into virions that were infectious. SIV239FV1b was sensitive to neutralization by MAb M2, with a 50% inhibitory concentration of 1 mug/ml. Neither SIV239FV2b nor SIV239FV4a was sensitive to M2 neutralization. The ability of the M2 antibody to neutralize SIV239FV1b infectivity was associated with an increased ability of the M2 antibody to detect native, oligomeric SIV239FV1b envelope protein on the surfaces of cells relative to that for the other SIV FLAG variants. Furthermore, SIV239FV1b was globally more sensitive to antibody-mediated neutralization than was parental SIV239 when these strains were screened with a panel of anti-SIV MAbs of various specificities. These results indicate that the V1 loop can serve as an effective target for neutralization on SIV239FV1b. However, antibody-mediated neutralization of this variant, similar to that of other SIV239 variants that have been studied previously, was associated with a global increase in neutralization sensitivity. These results suggest that the variable loops on the neutralization-resistant SIV239 strain are difficult for antibodies to access effectively and that mutations that allow neutralization have global effects on the trimeric envelope glycoprotein structure and accessibility.

Mode of inhibition of HIV-1 Integrase by a C-terminal domain-specific monoclonal antibody*

BackgroundTo further our understanding of the structure and function of HIV-1 integrase (IN) we developed and characterized a library of monoclonal antibodies (mAbs) directed against this protein. One of these antibodies, mAb33, which is specific for the C-terminal domain, was found to inhibit HIV-1 IN processing activity in vitro; a corresponding Fv fragment was able to inhibit HIV-1 integration in vivo. Our subsequent studies, using heteronuclear nuclear magnetic resonance spectroscopy, identified six solvent accessible residues on the surface of the C-terminal domain that were immobilized upon binding of the antibody, which were proposed to comprise the epitope. Here we test this hypothesis by measuring the affinity of mAb33 to HIV-1 proteins that contain Ala substitutions in each of these positions. To gain additional insight into the mode of inhibition we also measured the DNA binding capacity and enzymatic activities of the Ala substituted proteins.ResultsWe found that Ala substitution of any one of five of the putative epitope residues, F223, R224, Y226, I267, and I268, caused a decrease in the affinity of the mAb33 for HIV-1 IN, confirming the prediction from NMR data. Although IN derivatives with Ala substitutions in or near the mAb33 epitope exhibited decreased enzymatic activity, none of the epitope substitutions compromised DNA binding to full length HIV-1 IN, as measured by surface plasmon resonance spectroscopy. Two of these derivatives, IN (I276A) and IN (I267A/I268A), exhibited both increased DNA binding affinity and uncharacteristic dissociation kinetics; these proteins also exhibited non-specific nuclease activity. Results from these investigations are discussed in the context of current models for how the C-terminal domain interacts with substrate DNA.ConclusionIt is unlikely that inhibition of HIV-1 IN activity by mAb33 is caused by direct interaction with residues that are essential for substrate binding. Rather our findings are most consistent with a model whereby mAb33 binding distorts or constrains the structure of the C-terminal domain and/or blocks substrate binding indirectly. The DNA binding properties and non-specific nuclease activity of the I267A derivatives suggest that the C-terminal domain of IN normally plays an important role in aligning the viral DNA end for proper processing.

Highly Efficient Tandem Affinity Purification of Trypanosome Protein Complexes Based on a Novel Epitope Combination

Tandem affinity purification (TAP) allows for rapid and efficient purification of epitope-tagged protein complexes from crude extracts under native conditions. The method was established in yeast and has been successfully applied to other organisms, including mammals and trypanosomes. However, we found that the original method, which is based on the TAP tag, consisting of a duplicate protein A epitope, a tobacco etch virus protease cleavage site, and the calmodulin-binding peptide (CBP), did not yield enough recovery of transcription factor SNAPc (for small nuclear RNA-activating protein complex) from crude trypanosome extracts for protein identification. Specifically, the calmodulin affinity chromatography step proved to be inefficient. To overcome this problem, we replaced CBP by the protein C epitope (ProtC) and termed this new epitope combination PTP tag. ProtC binds with high affinity to the monoclonal antibody HPC4, which has the unique property of requiring calcium for antigen recognition. Thus, analogous to the calcium-dependent CBP-calmodulin interaction, ProtC-tagged proteins can be released from immobilized HPC4 by a chelator of divalent cations. While this property was retained, epitope substitution improved purification in our experiments by eliminating the inefficiency of calmodulin affinity chromatography and by providing an alternative way of elution using the ProtC peptide in cases where EGTA inactivated protein function. Furthermore, HPC4 allowed highly sensitive and specific detection of ProtC-tagged proteins after protease cleavage. Thus far, we have successfully purified and characterized the U1 small nuclear ribonucleoprotein particle, the transcription factor complex TATA-binding protein related factor 4 (TRF4)/SNAPc/transcription factor IIA (TFIIA), and RNA polymerase I of Trypanosoma brucei.

Investigation of the alternatively spliced insert region of the D 2L dopamine receptor by epitope substitution

Alternatively spliced variants of the D 2 dopamine receptor have distinct neuronal function and localization. The long isoform (D 2L) of this heptahelical transmembrane receptor differs from the short form only by the presence of a 29-amino acid insert in the third intracellular loop—a region known to be important for G protein coupling. Short and long isoforms have been shown to have distinct Gα i/o protein coupling specificities. However, the exact role of the alternatively spliced insert region in D 2 dopamine receptor function needs a more comprehensive examination. One way to address this is to substitute the entire insert region with an equivalent length, yet nonhomologous protein sequence. This report demonstrates the feasibility of replacing the 29-amino acid insert with a hemagglutinin double epitope tag with no recognizable functional consequences. The D 2L mutant is indistinguishable from the wild type D 2L receptor in terms of its ligand binding characteristics, as well as two effector responses: the agonist-mediated inhibition of forskolin-stimulated cAMP production, and agonist-stimulated MAPK phosphorylation. These data demonstrate that the epitope substitution generates a functional receptor, and that the alternatively spliced insert region, itself, does not appear to play a direct role in signal transduction. The epitope substitution permits dissection of sequence-mediated effects from structural effects due to the presence of the alternatively spliced insert region. Thus, this new construct could be a valuable tool for the study of D 2 receptor function.

Simian-human immunodeficiency virus escape from cytotoxic T-lymphocyte recognition at a structurally constrained epitope.

Virus-specific cytotoxic T lymphocytes (CTL) exert intense selection pressure on replicating simian immunodeficiency virus (SIV) and human immunodeficiency virus type 1 (HIV-1) in infected individuals. The immunodominant Mamu-A(*)01-restricted Gag p11C, C-M epitope is highly conserved among all sequenced isolates of SIV and therefore likely is structurally constrained. The strategies used by virus isolates to mutate away from an immunodominant epitope-specific CTL response are not well defined. Here we demonstrate that the emergence of a position 2 p11C, C-M epitope substitution (T47I) in a simian-human immunodeficiency virus (SHIV) strain 89.6P-infected Mamu-A(*)01(+) monkey is temporally correlated with the emergence of a flanking isoleucine-to-valine substitution at position 71 (I71V) of the capsid protein. An analysis of the SIV and HIV-2 sequences from the Los Alamos HIV Sequence Database revealed a significant association between any position 2 p11C, C-M epitope mutation and the I71V mutation. The T47I mutation alone is associated with significant decreases in viral protein expression, infectivity, and replication, and these deficiencies are restored to wild-type levels with the introduction of the flanking I71V mutation. Together, these data suggest that a compensatory mutation is selected for in SHIV strain 89.6P to facilitate the escape of that virus from CTL recognition of the dominant p11C, C-M epitope.

Mutational analysis of the adeno-associated virus type 2 (AAV2) capsid gene and construction of AAV2 vectors with altered tropism.

Adeno-associated virus type 2 (AAV2) has proven to be a valuable vector for gene therapy. Characterization of the functional domains of the AAV capsid proteins can facilitate our understanding of viral tissue tropism, immunoreactivity, viral entry, and DNA packaging, all of which are important issues for generating improved vectors. To obtain a comprehensive genetic map of the AAV capsid gene, we have constructed 93 mutants at 59 different positions in the AAV capsid gene by site-directed mutagenesis. Several types of mutants were studied, including epitope tag or ligand insertion mutants, alanine scanning mutants, and epitope substitution mutants. Analysis of these mutants revealed eight separate phenotypes. Infectious titers of the mutants revealed four classes. Class 1 mutants were viable, class 2 mutants were partially defective, class 3 mutants were temperature sensitive, and class 4 mutants were noninfectious. Further analysis revealed some of the defects in the class 2, 3, and 4 mutants. Among the class 4 mutants, a subset completely abolished capsid formation. These mutants were located predominantly, but not exclusively, in what are likely to be beta-barrel structures in the capsid protein VP3. Two of these mutants were insertions at the N and C termini of VP3, suggesting that both ends of VP3 play a role that is important for capsid assembly or stability. Several class 2 and 3 mutants produced capsids that were unstable during purification of viral particles. One mutant, R432A, made only empty capsids, presumably due to a defect in packaging viral DNA. Additionally, five mutants were defective in heparan binding, a step that is believed to be essential for viral entry. These were distributed into two amino acid clusters in what is likely to be a cell surface loop in the capsid protein VP3. The first cluster spanned amino acids 509 to 522; the second was between amino acids 561 and 591. In addition to the heparan binding clusters, hemagglutinin epitope tag insertions identified several other regions that were on the surface of the capsid. These included insertions at amino acids 1, 34, 138, 266, 447, 591, and 664. Positions 1 and 138 were the N termini of VP1 and VP2, respectively; position 34 was exclusively in VP1; the remaining surface positions were located in putative loop regions of VP3. The remaining mutants, most of them partially defective, were presumably defective in steps of viral entry that were not tested in the preliminary screening, including intracellular trafficking, viral uncoating, or coreceptor binding. Finally, in vitro experiments showed that insertion of the serpin receptor ligand in the N-terminal regions of VP1 or VP2 can change the tropism of AAV. Our results provide information on AAV capsid functional domains and are useful for future design of AAV vectors for targeting of specific tissues.

Mapping the Interaction Between Murine IgA and Murine Secretory Component Carrying Epitope Substitutions Reveals a Role of Domains II and III in Covalent Binding to IgA

We have identified sites for epitope insertion in the murine secretory component (SC) by replacing individual surface-exposed loops in domains I, II, and III with the FLAG sequence (Crottet, P., Peitsch, M. C., Servis, C., and Corthésy, B. (1999) J. Biol. Chem. 274, 31445-31455). We had previously shown that epitope-carrying SC reassociated with dimeric IgA (IgA(d)) can serve as a mucosal delivery vehicle. When analyzing the capacity of SC mutants to associate with IgA(d), we found that all domain II and III mutants bound specifically with immobilized IgA(d), and their affinity for IgA(d) was comparable to that of the wild type protein (IC(50) approximately 1 nM). We conclude that domains II and III in SC are permissive to local mutation and represent convenient sites to antigenize the SC molecule. No mutant bound to monomeric IgA. SC mutants exposing the FLAG at their surface maintained this property once bound to IgA(d), thereby defining regions not required for high affinity binding to IgA(d). Association of IgA(d) with SC mutants carrying a buried FLAG did not expose de novo the epitope, consistent with limited, local changes in the SC structure upon binding. Only wild type and two mutant SCs bound covalently to IgA(d), thus implicating domains II and III in the correct positioning of the reactive cysteine in SC. This establishes that the integrity of murine SC domains II and III is not essential to preserve specific IgA(d) binding but is necessary for covalency to take place. Finally, SC mutants existing in the monomeric and dimeric forms exhibited the same IgA(d) binding capacity as monomeric wild type SC known to bind with a 1:1 stoichiometry.