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. 2018 Jun 29;92(14):e00269-18.
doi: 10.1128/JVI.00269-18. Print 2018 Jul 15.

Breadth and Functionality of Varicella-Zoster Virus Glycoprotein-Specific Antibodies Identified after Zostavax Vaccination in Humans

Nicole L Sullivan  1   2 , Morgan A Reuter-Monslow  1 , Janet Sei  1 , Eberhard Durr  1 , Carl W Davis  2 , Cathy Chang  2 , Megan McCausland  2 , Andreas Wieland  2 , David Krah  3 , Nadine Rouphael  2   4 , Aneesh K Mehta  2   4 , Mark J Mulligan  2   4 , Bali Pulendran  2   5 , Rafi Ahmed  6 , Kalpit A Vora  7
Affiliations

Breadth and Functionality of Varicella-Zoster Virus Glycoprotein-Specific Antibodies Identified after Zostavax Vaccination in Humans

Nicole L Sullivan et al. J Virol. .

Abstract

Herpes zoster (HZ) (shingles) is the clinical manifestation of varicella-zoster virus (VZV) reactivation. HZ typically develops as people age, due to decreased cell-mediated immunity. However, the importance of antibodies for immunity against HZ prevention remains to be understood. The goal of this study was to examine the breadth and functionality of VZV-specific antibodies after vaccination with a live attenuated HZ vaccine (Zostavax). Direct enumeration of VZV-specific antibody-secreting cells (ASCs) via enzyme-linked immunosorbent spot assay (ELISPOT assay) showed that Zostavax can induce both IgG and IgA ASCs 7 days after vaccination but not IgM ASCs. The VZV-specific ASCs range from 33 to 55% of the total IgG ASCs. Twenty-five human VZV-specific monoclonal antibodies (MAbs) were cloned and characterized from single-cell-sorted ASCs of five subjects (>60 years old) who received Zostavax. These MAbs had an average of ∼20 somatic hypermutations per VH gene, similar to those seen after seasonal influenza vaccination. Fifteen of the 25 MAbs were gE specific, whereas the remaining MAbs were gB, gH, or gI specific. The most potent neutralizing antibodies were gH specific and were also able to inhibit cell-to-cell spread of the virus in vitro Most gE-specific MAbs were able to neutralize VZV, but they required the presence of complement and were unable to block cell-to-cell spread. These data indicate that Zostavax induces a memory B cell recall response characterized by anti-gE > anti-gI > anti-gB > anti-gH antibodies. While antibodies to gH could be involved in limiting the spread of VZV upon reactivation, the contribution of anti-gE antibodies toward protective immunity after Zostavax needs further evaluation.IMPORTANCE Varicella-zoster virus (VZV) is the causative agent of chickenpox and shingles. Following infection with VZV, the virus becomes latent and resides in nerve cells. Age-related declines in immunity/immunosuppression can result in reactivation of this latent virus, causing shingles. It has been shown that waning T cell immunity correlates with an increased incidence of VZV reactivation. Interestingly, serum with high levels of VZV-specific antibodies (VariZIG; IV immunoglobulin) has been administered to high-risk populations, e.g., immunocompromised children, newborns, and pregnant women, after exposure to VZV and has shown some protection against chickenpox. However, the relative contribution of antibodies against individual surface glycoproteins toward protection from shingles in elderly/immunocompromised individuals has not been established. Here, we examined the breadth and functionality of VZV-specific antibodies after vaccination with the live attenuated VZV vaccine Zostavax in humans. This study will add to our understanding of the role of antibodies in protection against shingles.

Keywords: B cell; VZV; VZV glycoprotein; Zostavax; antibody; cloning; herpes zoster; immunology; shingles.

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Figures

FIG 1
FIG 1
Kinetics of VZV-specific antibody-secreting cells (ASCs) after Zostavax vaccination in older adults. (A) Representative VZV-specific (top row) and total (bottom row) IgG (left), IgM (center), and IgA (right) ASCs were measured on days 0, 7, and 14 after Zostavax vaccination via ELISPOT assay. (B) Cumulative results for VZV-specific IgG, IgM, and IgA ASCs after Zostavax vaccination. *, P < 0.05 by the Student t test.
FIG 2
FIG 2
ASC sorting and frequency of VZV-specific ASCs. Five older adults were vaccinated with Zostavax, and PBMCs were collected on day 7. (A) ASCs were single-cell sorted into PCR plates using a FACSAria II (left and middle panels, presort; right panel, postsort to assess purity). (B) The frequency of VZV-specific ASCs was calculated via IgG ELISPOT assay. Top row, VZV-specific IgG ASCs; bottom row, total IgG ASCs. The percentages of specific ASCs were measured by taking the number of VZV-specific IgG ASCs divided by the total number of IgG ASCs detected. The number of ASCs plated per well is listed.
FIG 3
FIG 3
Comparison of the numbers of VH somatic hypermutations in naive B cells, ASCs sorted after vaccination with the 2009/10 influenza trivalent vaccine, and ASCs after Zostavax vaccination. Data from the 2009/10 influenza trivalent vaccine were published by Li et al. (50). IgBLAST was used to determine the number of somatic hypermutations (Fr1-CDR3 heavy chain) as described previously (49).
FIG 4
FIG 4
Work flow to identify glycoprotein-specific VZV monoclonal antibodies. Supernatants generated using ASCs single cell sorted, cloned, and expressed in Expi293 cells were tested for VZV lysate binding via ELISA. VZV glycoprotein-positive but non-gE antibodies (10/25) were tested via immunoprecipitation followed by LC/MS to determine the specificity. All glycoprotein-positive antibodies were then tested for neutralization and cell-to-cell spread inhibition in vitro.
FIG 5
FIG 5
Identification of non-gE-specific monoclonal antibodies. Immunoprecipitation followed by nano-LC-MS/MS was done using the gP-specific monoclonal antibodies and the top three MS hits listed. (A) VZV gB-specific monoclonal antibodies. (B) VZV gH-specific monoclonal antibodies. (C) VZV gI-specific monoclonal antibodies. (D) Western blotting with RM-1D1, 302-1B12, 302-1C12, 303-1C1, and 303-1B2 antibodies was performed using a glycoprotein lysate (including all VZV glycoproteins). The table shows the predicted molecular weights and pIs. (E) Of the 25 VZV glycoprotein-positive antibodies, 15 were gE specific, 5 were gI specific, 3 were gB specific, and 2 were gH specific.
FIG 6
FIG 6
In vitro VZV neutralization. Purified VZV-specific monoclonal antibodies were tested for in vitro neutralization as described in Materials and Methods. Using an Acumen Cellista, the total number of cells expressing GFP per well was quantified. (A) Cumulative results with and without complement. (B) Cumulative IC50 results (in μg/ml) for all antibodies that showed neutralization with or without the addition of guinea pig complement. (C) Representative neutralization plots with gH-, gE-, and gI-specific monoclonal antibodies added. Red, guinea pig complement added; blue, without guinea pig complement.
FIG 7
FIG 7
In vitro cell-to-cell spread inhibition. Cell-to-cell spread inhibition was performed as described in Materials and Methods. (A) Representative well images for samples treated with complement. PGS buffer was used as a negative control. (B) Cumulative data for anti-gE antibodies (top) and non-gE antibodies (bottom). Black bars represent antibodies added at 10 μg/ml, and green bars represent the same antibodies added at 1 μg/ml. (C) Cell spread inhibition curves with IC50 values (μg/ml) with anti-gH or irrelevant MAbs, with (left) or without (right) complement added.
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