Unusual Features of Vaccinia Virus Extracellular Virion Form Neutralization Resistance Revealed in Human Antibody Responses to the Smallpox Vaccine

  1. Shane Crottya
  1. aDivision of Vaccine Discovery
  2. bDivision of Cell Biology, La Jolla Institute for Allergy and Immunology (LIAI), La Jolla, California, USA
  3. cVanderbilt Vaccine Center, Vanderbilt University Medical Center, Nashville, Tennessee, USA
  4. dDepartment of Immunology and Microbial Science and IAVI Neutralizing Antibody Center, The Scripps Research Institute, La Jolla, California, USA
  5. eDNA Array Core Facility and Consortium for Functional Glycomics, The Scripps Research Institute, La Jolla, California, USA
  6. fDivision of Infectious Diseases, Department of Medicine, University of California, Irvine, California, USA
  7. gKyowa Hakko Kirin California, La Jolla, California, USA
  8. hDepartment of Microbiology and Immunology, University of Texas Health Science Center, San Antonio, Texas, USA
  1. Fig 1
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    Fig 1

    Human anti-B5 antibodies are not the only specificities mediating EV neutralization. VACV EV neutralization activity of plasma samples in the absence of complement (A) or the presence of complement (C′) (B). (C and D) Quantitation of anti-B5 IgG in untreated plasma (C) or in plasma after blocking with 10 μg of rB5 protein (D) from vaccinated human donors (lanes a to i) or a nonimmune human donor (Non imm). An IgG signal level of 2 relative units (RU) was selected as a stringent cutoff (dashed line), establishing 98% specificity (see Materials and Methods). (E) VACV viral protein microarrays. B5 protein spots (red box) in untreated plasma from an individual immunized with the smallpox vaccine (human plasma, left) and B5-treated plasma (human plasma + rB5, right). B5 protein is also present in row 12, spot 4. The C3L spot was not printed on the left panel, row 11, spot 8. (F) VACV EV neutralization activity after rB5 blockade of anti-B5 MAb h101 at 10 μg/ml. (G) VACV EV neutralization activity of plasma samples after the blockade of anti-B5 antibodies in the presence of complement. VACV EV alone (A) or plus complement (B and G) were negative controls (—). Dashed lines in panels A, B, F, and G indicate 50% of the plaque numbers of VACV EV with or without complement. *, P < 0.005; **, P < 0.0004; ***, P < 0.0001. Error bars indicate SEM under each condition. All data are representative of two or more experiments. (H) Quantitation of anti-A33 binding IgG in plasma from donors by recombinant A33 protein (rA33) ELISA. Data are representative of two independent experiments. OD, optical density. An IgG signal level of 0.2 was selected as the cutoff (dashed line).

  2. Fig 2
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    Fig 2

    Complement and isotype dependence of murine anti-A33 MAb neutralization of VACV EV. (A to C) VACV EV neutralization by anti-A33 rabbit PAbs is dependent on complement. (A) VACV EV neutralization activity of rabbit PAbs against A33 (NR628) at 1/100 dilution in the absence or the presence of complement. VACV EV alone and a naïve rabbit serum (rabbit serum) were negative controls. (B) Titrated VACV EV neutralization activity of NR628 in the absence (closed circle) or the presence (open circles) of complement. Negative-control samples are shown at single dilutions: naïve rabbit serum alone (closed squares) or plus complement (open squares). The dashed line indicates the plaque numbers of VACV EV in the presence of complement without antibody. (C) VACV EV neutralization by anti-A33 PAb is independent of anti-MV Abs. VACV EV neutralization activity of NR628 at 1/100 dilution with or without complement in the absence of anti-L1 Ab. VACV EV with and without complement (—) were negative controls. (D) Rabbit anti-A33 PAb exhibited comet tail inhibition activity in vitro. Absence of Ab, no treatment; N628, anti-A33 PAb; naïve rabbit serum, rabbit serum. Data in panels B and D are representative of two independent experiments. (E) Sequence analysis of heavy- and light-chain variable regions of murine anti-A33 MAbs (12D4.1 and KA10). Framework (FR) and CDR regions are shown. (F and G) VACV EV neutralization is dependent on complement and anti-A33 MAb isotype. (F) Neutralization activity of purified murine anti-A33 MAbs (12D4.1, KA10, and NR565) in the presence of anti-L1 Abs and in the absence (left) or the presence (right) of complement. Murine anti-B5 MAbs B126 (IgG2a) and B96 (IgG1) were used as controls for neutralization activity in each panel. Anti-DNP (IgG1) and VACV EV with or without complement (—) were negative controls. (G) VACV EV neutralization activity of mouse anti-A33 MAb IgG1 isotype (NR565) in the absence (left) or the presence (middle) of complement-fixing anti-mouse PAb (2° Ab IgG) with or without complement. Murine anti-B5 MAb B126 (IgG2a) was used as a positive control. Human anti-DNP MAb (isotype control) at 10 μg/ml with or without 2° Ab IgG with or without complement was used as a negative control (right). 2° Ab IgG with or without complement also was used as a negative control. Error bars indicate SEM under each condition. The dashed line indicates 50% of the plaque numbers of VACV EV with or without complement in panels A, C, F, and G. All data are representative of three or more experiments.

  3. Fig 3
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    Fig 3

    Neutralization of EV by human anti-A33 MAb with effector function. (A) Titration of fully human anti-A33 IgG1 MAbs (VV22 and VV80) by rA33 ELISA. Human anti-DNP was used as a negative control. OD, optical density. (B) Cell surface expression of A33 on VACV-B5-GFP-infected cells detected by human anti-A33 MAbs VV22 (red line curve) and VV80 (green line curve) using flow cytometry. Anti-DNP MAb was the negative control (gray line curve). Data are representative of three independent experiments. (C) Sequence analysis of heavy-chain variable regions of human anti-A33 MAbs (VV22 and VV80). Framework (FR) and CDR regions are shown. (D and E) VACV EV virion neutralization by fully human anti-A33 MAbs. (D) VACV EV neutralization activity of human anti-A33 isotype IgG1 MAbs (VV22 and VV80 clones) in the presence of anti-L1 Abs with or without complement. (E) Titrated VACV EV neutralization activity of human anti-A33 MAbs VV22 (closed squares) and VV80 (open circles) in the presence of anti-L1 Abs with or without complement. Human anti-DNP MAb (open squares) and EV VACV (closed diamonds) with or without complement were used as negative controls. Data in panel D are representative of two experiments, each of which was done in the presence of anti-L1. Data are represented as plaque numbers. (F) VACV EV neutralization activity of human anti-A33 MAbs with or without complement and in the absence of anti-L1 Abs. Human anti-B5 MAb clone h101 (IgG1) was used as the positive control in each experiment. Human anti-DNP MAb (IgG1) and VACV EV with or without complement (—) were used as a negative control. The dashed line indicates 50% of the plaque numbers of VACV EV with or without complement in panels D and F. Error bars indicate SEM under each condition. All data in panels D and F are representative of three or more experiments.

  4. Fig 4
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    Fig 4

    Combined effect of human A33 and B5 against EV virion and the impact of anti-A33 Ab in smallpox-vaccinated humans. (A) VACV EV neutralization activity of the fully human MAbs against B5 (h102) and A33 (VV22) at 20, 10, or 1 μg/ml of each MAb in the absence or presence of 1% complement. (B) Irrelevant human IgG1 MAb and EV (anti-DNP) at 40, 20, or 2 μg/ml were negative controls with or without complement. Data are represented as plaque numbers. The dashed line indicates 50% of the plaque numbers of VACV EV with or without complement. (C) VACV EV neutralization activity (%) of plasma from donors (b, c, e, g, and i) in the presence of complement and after the blockade of anti-A33 or anti-B5/A33 Abs with 10 μg of rA33 or rB5/rA33 proteins. *, P < 0.05. The dashed line indicates 50% of neutralization activity. Error bars indicate SEM under each condition. All data are representative of two independent experiments.

  5. Fig 5
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    Fig 5

    Human anti-A56 MAbs have no effect on EV neutralization. (A to C) Vero E6 monolayers cell were infected with VACV-B5-GFP (green), and surface expression of A56 (red) was determined 12 h postinfection by surface staining with human anti-A56 MAb ES1 or WR2 and performing immunofluorescence (A) or flow cytometry (B and C). Surface expression of A56 was tested after infection with VACV-B5-GFP by surface staining infected cells with human anti-A56 MAb ES1 (red curve) or anti-DNP MAb (control; gray curve). (C) MFI of cell-based ELISA, quantitating surface-bound anti-A56 MAbs to VACV infected cells. Data are representative of three independent experiments. Irrelevant human MAb (anti-DNP, IgG1) was used as a negative control. (D) Sequence analysis of heavy-chain variable regions of human anti-A56 MAbs (WR2 and ES1). Framework (FR) and CDR regions are shown. (E) Human anti-A56 MAbs do not neutralize VACV EV. VACV EV neutralization activity of human anti-A56 MAbs (ES1 and WR2) with or without complement in the presence of anti-L1 Abs. Complement-fixing human anti-B5 MAb h101 (IgG1) was used as a positive control in each experiment. (F) Comet tail plaque inhibition. Shown are the absence of Ab (termed no treatment) or presence of anti-A56 MAbs (ES1 and WR2) or irrelevant MAb (control IgG1). Data are representative of two independent experiments. (G) Addition of complement-fixing anti-human IgG at 10 μg/ml (left) to human anti-A56 MAbs does not improve the neutralization of VACV EV. VACV EV neutralization activity of mouse anti-A33 MAb (NR565) in the absence or presence of complement-fixing anti-mouse IgG at 10 μg/ml with and without complement were control Abs (right). Irrelevant human MAb (anti-DNP, IgG1) was used as a negative control (right). The dashed line indicates 50% of the plaque numbers of VACV EV with or without complement. Error bars indicate SEM under each condition. All data in panels E and G are representative of three or more experiments.

  6. Fig 6
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    Fig 6

    Expanding the footprint of single-specificity MAb does not result in EV neutralization. (A to C) VACV EV neutralization activity of mouse anti-B5 MAbs (B96, IgG1), mouse anti-A33 MAbs (NR565, IgG1), or human anti-A56 MAbs (ES1 or WR2, IgG1) at 10 μg/ml alone (A), in the presence of species-specific anti-Ig secondary antibodies (B), or in the presence of secondary and tertiary anti-Ig antibodies (C). EV alone (—) and human anti-DNP IgG1 (“IgG1”) were negative controls. Data are representative of two independent experiments. The dashed line indicates 50% of plaque numbers of VACV EV with or without complement. Error bars indicate SEM under each condition.

  7. Fig 7
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    Fig 7

    Simultaneously coating EV virion with human MAbs specific to multiple antigens, B5, A33, and A56, is insufficient for neutralization of EV from the WR or IHD-J strain. VACV EV neutralization activity of human anti-A33 (VV22), A56 (WR2), and B5 (h101) IgG1 MAbs, starting at 20 μg/ml of each MAb and then using 2-fold serial dilutions, against VACV EV of the WR (A) or IHD-J strain (I) with or without a small amount of complement (1%) was determined. (B and J) Irrelevant human MAb (anti-DNP, control IgG1), starting at 60 μg/ml and then 2-fold serial dilution, in the presence of VACV EV WR (B) or IHD-J strain (J) with or without complement (1%). All data are representative of three or more experiments. (C to F) VACV EV neutralization activity of human anti-A33 (VV22) and B5 (h101) MAbs IgG1 (C) in combination with human anti-A56 (WR2) (E), starting at 40 μg/ml of each MAb, against VACV EV of WR with or without complement (1%). (D and F) Irrelevant human MAb (anti-DNP; control IgG1), starting at 80 or 120 μg/ml, in the presence of VACV EV with or without complement (1%). The data are representative of one independent experiment. (G and H) VACV EV neutralization activity of human anti-A56 MAbs in the presence of anti-L1 Abs against the VACVWR (G) or VACVIHDJ strain (H) with or without complement (10%) in each experiment. Mouse anti-B5 MAb B126 (IgG2a) and rabbit anti-A33 PAbs (NR628) were used as positive controls. VACV EV alone (—) and an irrelevant human IgG1 MAb plus EV (IgG1) were negative controls. The dashed line indicates 50% of the plaque numbers of VACV EV with or without complement. Error bars indicate SEM under each condition. All data are representative of two independent experiments.

  8. Fig 8
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    Fig 8

    Mechanism of EV neutralization. (A) Models of VACV EV neutralization. Schematic diagrams of potential virion neutralization pathways. B5, A33, and A56 are drawn in green, blue, and gray, respectively. An unknown hypothetical EV surface antigen is drawn in cyan. Abs in model I are anti-B5, anti-A33, and anti-A56. Abs in model II are anti-B5 and anti-A33. Complement (C′) components include C1q and C3. Model I is the basic occupancy model. MAbs against three exposed EV antigens, B5, A33, and A56, could completely coat the functional antigens on the virion surface and subsequently neutralize the virus. This model failed. Direct occupancy of B5, A33, and A56 with Abs is insufficient to block infection of targets cells. VACV EV escape of neutralization by anti-B5, anti-A33, and anti-A56 Ab binding may be due to limited abundance of each antigen on the surface of EV, resulting in a lack of blocking of an unknown EV surface protein still able to facilitate infection. Alternatively, the infectious component of the virion surface is lipid and there is insufficient viral protein on the surface for the anti-VACV Abs to cover the surface, leaving the fusogenic membrane accessible. Model II shows complement-assisted single-specificity or two-specificity anti-EV MAbs coating VACV EV via opsonization. Antibody-mediated protection against VACV EV is dependent on activation of complement C1q via the Fc domain of the immunoglobulin and covalent attachment of C3 to the lipid outer membrane of the virus. (B and C) The presence of anti-MV Abs in EV stock is not necessary for EV neutralization. Schematic diagrams of potential virion neutralization pathways. EV and damaged EV (EV/MV) particles are presented in EV stock. B5 and A33 are drawn in cyan and gray, respectively. L1 or H3, as an MV protein, is drawn in green. The Abs were anti-B5 and anti-A33. Complement (C′) components included C1q and C3. (B) MAbs against one or two exposed EV antigens, B5 and/or A33, could completely coat the functional antigens on EV and EV/MV and subsequently neutralize EV in the presence of complement components C1q and C3. (C) MAbs against one or two exposed EV antigens, B5 and/or A33, could completely coat the specific antigens on EV and EV/MV, and anti-MV Abs coat a specific antigen on the surface of EV/MV particles. Subsequently, the addition of the complement enhances the neutralization of EV and EV/MV particles.

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  1. Accepted manuscript posted online 14 November 2012, doi: 10.1128/JVI.02152-12 J. Virol. vol. 87 no. 3 1569-1585
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