Protection from cytomegalovirus viremia following glycoprotein B vaccination is not dependent on neutralizing antibodies - PubMed (original) (raw)

Protection from cytomegalovirus viremia following glycoprotein B vaccination is not dependent on neutralizing antibodies

Ilona Baraniak et al. Proc Natl Acad Sci U S A. 2018.

Abstract

Human cytomegalovirus (HCMV) is an important pathogen in transplant patients and in congenital infection. Previously, we demonstrated that vaccination with a recombinant viral glycoprotein B (gB)/MF59 adjuvant formulation before solid organ transplant reduced viral load parameters post transplant. Reduced posttransplant viremia was directly correlated with antibody titers against gB consistent with a humoral response against gB being important. Here we show that sera from the vaccinated seronegative patients displayed little evidence of a neutralizing antibody response against cell-free HCMV in vitro. Additionally, sera from seronegative vaccine recipients had minimal effect on the replication of a strain of HCMV engineered to be cell-associated in a viral spread assay. Furthermore, although natural infection can induce antibody-dependent cellular cytotoxicity (ADCC) responses, serological analysis of seronegative vaccinees again presented no evidence of a substantial ADCC-promoting antibody response being generated de novo. Finally, analyses for responses against major antigenic domains of gB following vaccination were variable, and their pattern was distinct compared with natural infection. Taken together, these data argue that the protective effect elicited by the gB vaccine is via a mechanism of action in seronegative vaccinees that cannot be explained by neutralization or the induction of ADCC. More generally, these data, which are derived from a human challenge model that demonstrated that the gB vaccine is protective, highlight the need for more sophisticated analyses of new HCMV vaccines over and above the quantification of an ability to induce potent neutralizing antibody responses in vitro.

Keywords: cytomegalovirus; humoral immunity; vaccine.

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Conflict of interest statement

Conflict of interest statement: Sylvie Pichon is an employee of Sanofi Pasteur. However, there are no personal financial gains regarding the publication of this manuscript.

Figures

Fig. 1.

Fig. 1.

Vaccination does not promote neutralizing antibody responses in seronegatives. (A) HCMV was incubated for 1 h with different concentrations of monoclonal antibodies against gB (ITC88 and 2F12), IgG1 isotype control, or media and then used to infect HFFs (MOI = 0.5). Percentage of IE positivity was scored 24 hpi. n = 3. (B) HCMV was incubated for 1 h with sera from seropositive or seronegative patients either vaccinated with gB or given a placebo and then used to infect HFFs (MOI = 1). The analysis was performed on sera isolated prevaccination and at day of transplant (post vaccination). Percentage IE positivity was scored 24 hpi and further stratified into patients who developed viremia. n = 3. (C) HCMV was incubated for 1 h with sera from seronegative patients either vaccinated with gB or given placebo and then used to infect HFFs (MOI = 1). The analysis was performed on sera isolated prevaccination and at day of transplant (post vaccination). Percentage of pp28 positivity was scored 120 hpi and further stratified into patients who developed viremia. n = 3.

Fig. 2.

Fig. 2.

Sera from vaccinated seronegatives does not control the spread of cell-associated HCMV in vitro. (A) HFFs were infected with Merlin-IE2-GFP (MOI = 0.01), and the progress of infection was monitored for 2 wk. Representative images of GFP expression at 1, 6, 9, and 14 d post infection are shown. Cells were counterstained with DAPI to show cell layers. (B and C) HFFs infected with Merlin-IE2-GFP (B) or Merlin (C) were incubated 24 hpi with ITC88 (100 μg/mL) and viral spread assay 2 wk post infection. GFP (cell-associated) or IE immunostaining (cell-free) was used to calculate the percentage of infection. n = 3. (D) HFFs infected with Merlin (cell free) or Merlin-IE2-GFP (cell-associated) were incubated 24 hpi with no sera (control), seropositive sera, or seronegative sera. After 2 wk, GFP (cell-associated) or IE immunostaining (cell-free) was used to calculate the percentage of infection. n = 3. (E) HFFs infected with Merlin-IE2-GFP (cell-associated) were incubated 24 hpi with no sera (infected cells), healthy donor seropositive [HCMV(+)] sera, or seronegative [HCMV(−)] sera. Alternatively, HFFs were incubated with sera from either seropositive or seronegative patients given gB vaccine or placebo. Sera prevaccination and at day of transplant (post vaccination) were analyzed. After 2 wk, GFP (cell-associated) was used to calculate the percentage of infection. n = 3. Patients were further stratified into those who experienced viremia versus those who did not.

Fig. 3.

Fig. 3.

Increased ADCC antibody responses against gB are not detected in seropositives. (A) Gating strategy to study evidence of ADCC activity. NK cells defined as CD56+CD3− were then assayed for CD107a expression or IFNg. PMA/Ionomycin was used as a positive control. (B) Titration of healthy donor sera from seropositive and seronegative donors for ability to promote CD107a expression on NK cells. (C and D) Summary of data of ADCC responses in seropositive liver and kidney organ recipients at time of transplant. Comparisons between placebo and vaccination or between viremia or no viremia shown. n = 3.

Fig. 4.

Fig. 4.

Vaccination does not induce detectable ADCC antibody responses against gB in seronegatives. (A_–_D) Longitudinal sera samples from multiple visits were analyzed for ADCC-promoting activity. Samples were prevaccination (v#1) or 1 (v#2), 2 (v#3), 6 (v#4), and 7 (v#5) mo post vaccination or at time of transplant (d0) or 7 d post transplant (d7). Baseline negative controls are shown using unstimulated cells or healthy donor seronegative sera, and PMA/Ionomycin served as positive control.

Fig. 5.

Fig. 5.

Vaccination induces a pattern of epitope responses distinct from natural infection. (A_–_H) ELISAs were performed on sera prevaccination (0 mo) or 1, 2, 6, and 7 mo post vaccination. Prevaccination represents background. ELISA ODs for anti-AD1 (A), AD2 (C), AD4 (E), and AD5 (G) responses are shown. Alternatively, data were stratified using outcome post transplant (B, D, F, and H) to assess impact of responses on viremia. Statistical significance was measured using a nonparametric Mann–Whitney U test. ns, nonsignificant.

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